Detecting CYP24 expression level as a marker for predisposition to cancer

ABSTRACT

This invention pertains to the discovery that an amplification of the CYP24 gene or an increase in CYP24 activity is a marker for the presence of, progression of, or predisposition to, a cancer (e.g., breast cancer). Using this information, this invention provides methods of detecting a predisposition to cancer in an animal. The methods involve (i) providing a biological sample from an animal (e.g. a human patient); (ii) detecting the level of CYP24 within the biological sample; and (iii) comparing the level of CYP24 with a level of CYP24 in a control sample taken from a normal, cancer-free tissue where an increased level of CYP24 in the biological sample compared to the level of CYP24 in the control sample indicates the presence of said cancer in said animal.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/633,643, filed Dec. 8, 2009, now U.S. Pat. No. 8,173,602, which is adivisional of U.S. application Ser. No. 09/285,292, filed Apr. 2, 1999,now U.S. Pat. No. 7,648,826.

This invention was made with Government support under Grant No. CA58207, awarded by the National Institutes of Health. The Government ofthe United States of America may have certain rights in this invention.

FIELD OF THE INVENTION

This invention pertains to the field of cancer genetics andcytogenetics. In particular, this invention pertains to theidentification of an association between amplification(s) of the CYP24gene and cancer.

BACKGROUND OF THE INVENTION

Chromosome abnormalities are often associated with genetic disorders,degenerative diseases, and cancer. The deletion or multiplication ofcopies of whole chromosomes and the deletion or amplifications ofchromosomal segments or specific regions are common occurrences incancer (Smith (1991) Breast Cancer Res. Treat. 18: Suppl. 1:5-14; van deVijer (1991) Biochim. Biophys. Acta. 1072:33-50). In fact,amplifications and deletions of DNA sequences can be the cause of acancer. For example, proto-oncogenes and tumor-suppressor genes,respectively, are frequently characteristic of tumorigenesis (Dutrillaux(1990) Cancer Genet. Cytogenet. 49: 203-217). Clearly, theidentification and cloning of specific genomic regions associated withcancer is crucial both to the study of tumorigenesis and in developingbetter means of diagnosis and prognosis.

Studies using comparative genomic hybridization (CGH) have revealedapproximately twenty amplified genomic regions in human breast tumors(Muleris (1994) Genes Chromosomes Cancer 10:160-170; Kalliioniemi (1994)Proc. Natl. Acad. Sci. USA 91: 2156-2160; Isola (1995) Am. J. Pathol.147:905-911). These regions are predicted to encode dominantly actinggenes that may play a role in tumor progression or response to therapy.Three of these amplified regions have been associated with establishedoncogenes: ERBB2 at 17q12, MYC at 8q24 and CCND1 and EMS1 at 11q13. Inbreast cancer, ERBB2 and CCND1/EMS1 amplification and overexpression areassociated with decreased life expectancy (Gaudray (1992) Mutat. Res.276:317-328; Borg (1991) Oncogene 6:137-143). MYC amplification has beenassociated with lymph node involvement, advanced stage cancer and anincreased rate of relapse (Borg (1992) Intern. J. Cancer 51: 687-691;Berns (1995) Gene 159: 11-18). Clearly, the identification of additionalamplified genomic regions associated with breast cancer or other tumorcells is critical to the study of tumorigenesis and in the developmentof cancer diagnostics.

One of the amplified regions found in the CGH studies was on chromosome20, specifically, 20q13. Amplification of 20q13 was subsequently foundto occur in a variety of tumor types and to be associated withaggressive tumor behavior. Increased 20q13 copy number was found in 40%of breast cancer cell lines and 18% of primary breast tumors(Kalliioniemi (1994) supra). Copy number gains at 20q13 have also beenreported in greater than 25% of cancers of the ovary (Iwabuchi (1995)Cancer Res. 55:6172-6180), colon (Schlegel (1995) Cancer Res. 55:6002-6005), head-and-neck (Bockmuhl (1996) Laryngor. 75: 408-414), brain(Mohapatra (1995) Genes Chromosomes Cancer 13: 86-93), and pancreas(Solinas-Toldo (1996) Genes Chromosomes Cancer 20:399-407).

The 20q13 region was analyzed at higher resolution in breast tumors andcell lines using fluorescent in situ hybridization (FISH). A 1.5megabase (Mb) wide amplified region within 20q13 was identified (Stokke(1995) Genomics 26: 134-137); Tanner (1994) Cancer Res. 54:4257-4260).Interphase FISH revealed low-level (>1.5×) and high level (>3×) 20q13sequence amplification in 29% and 7% of breast cancers, respectively(Tanner (1995) Clin. Cancer Res. 1: 1455-1461). High level amplificationwas associated with an aggressive tumor phenotype (Tanner (1995) supra;Courjal (1996) Br. J. Cancer 74: 1984). Another study, using FISH toanalyze 14 loci along chromosome 20q in 146 uncultured breastcarcinomas, identified three independently amplified regions, includingRMC20C001 region at 20q13.2 (highly amplified in 9.6% of the cases),PTPN1 region 3 Mb proximal (6.2%), and AIB3 region at 20q11 (6.2%)(Tanner (1996) Cancer Res. 56:3441-3445). Clearly, definitivecharacterization of amplified regions within 20q13 would be an importantstep in the diagnosis and prognosis of these cancers.

Increased copy number of chromosome 20q in cultured cells also has beenassociated with phenotypes characteristic of progressing tumors,including immortalization and genomic instability. For example,increased copy number at 20q11-qter has been observed frequently inhuman uro-epithelial cells (HUC) (Reznikoff (1994) Genes Dev. 8:2227-2240) and keratinocytes (Solinas-Toldo (1997) Proc. Natl. Acad.Sci. USA 94:3854-3859) after transfection with human papilloma virus(HPV)16 E7 or HPV16, respectively. In addition, increased copy number at20q13.2 has been associated with p53 independent genomic instability insome HPV16 E7 transfected HUC lines (Savelieva (1997) Oncogene 14:551-560). These studies suggest that increased expression of one or moregenes on 20q and especially at 20q13.2 contribute to the evolution ofbreast cancer and other solid tumors. Several candidate oncogenes havebeen identified as amplified on 20q, including AIB1 (Anzick (1997)Science 277: 965-968), BTAK (Sen (1997) Oncogene 14: 2195-200), CAS(Brinkmann (1996) Genome Res. 6: 187-194) and TFAP2C (Williamson (1996)Genomics 35:262-264). Clearly, definitive characterization of nucleicacid sequences in 20q13 associated with tumor phenotypes would be animportant step in the diagnosis and prognosis of these cancers. Thepresent invention fulfills these and other needs.

SUMMARY OF THE INVENTION

This invention pertains to the discovery that an amplification of theCYP24 gene or an increase in CYP24 activity is a marker for the presenceof, progression of, or predisposition to, a cancer (e.g., breastcancer). Using this information, this invention provides methods ofdetecting/evaluating a predisposition to, progression of, or prognosisof cancer in an animal. Thus, in one embodiment, this invention providesmethods of detecting a predisposition to cancer in an animal. Themethods involve providing a biological sample from said animal,detecting the level of CYP24 within the biological sample; and comparingthe level of CYP24 with a level of CYP24 in a control sample taken froma normal, cancer-free tissue;

-   -   where an increased level of CYP24 in the biological sample        compared to the level of CYP24 in the control sample indicates        the presence of a cancer in the animal. Similarly, an increased        level of CYP24 in the sample can indicate a poor prognosis for        an animal/patient known to have cancer, and/or a reduced        survival expectancy, and/or the actual presence of a cancer.

In one embodiment, the level of CYP24 is detected by determining thecopy number of CYP24 genes in the cells of the biological sample. In aparticularly preferred embodiment, the copy number is measured usingComparative Genomic Hybridization (CGH). In another preferredembodiment, the copy number is determined by hybridization to an arrayof nucleic acid probes and in another particularly preferred embodiment,the Comparative Genomic Hybridization is performed on an array.

In another embodiment, the level of CYP24 is detected by measuring thelevel of CYP24 mRNA in the biological sample (e.g., by hybridization toone or more probes in an array), wherein an increased level of CYP24 RNAin said sample compared to CYP24 RNA in said control sample indicates apredisposition to cancer. In preferred embodiments, the level of CYP24is measured in said biological sample and said control sample at thesame vitamin D receptor activity or the CYP24 levels are normalized tothe level of vitamin D receptor activity in the sample and control.

In still another embodiment, the level of CYP24 is detected by measuringthe level of CYP24 protein in the biological sample, where an increasedlevel of CYP24 protein in the sample as compared to CYP24 protein insaid control sample indicates a predisposition to cancer. In preferredembodiments, the level of CYP24 protein is measured in the biologicalsample and the control sample at the same vitamin D receptor activity orthe protein levels are normalized to the level of vitamin D receptoractivity in the sample and control.

In still yet another embodiment, the level of CYP24 is detected bymeasuring the level of 25-hydroxyvitamin D3 24-hydroxylase enzymeactivity in the biological sample, wherein an increased level of25-hydroxyvitamin D3 24-hydroxylase enzyme activity in the sample ascompared to 25-hydroxyvitamin D3 24-hydroxylase enzyme activity in thecontrol sample indicates a predisposition to cancer. In preferredmethods, the level of 25-hydroxyvitamin D3 24-hydroxylase activity ismeasured in the biological sample and the control sample at the samevitamin D receptor activity or the activity levels are normalized to thelevel of vitamin D receptor activity in the sample and control.

In the methods described herein, the animal(s) are mammals, morepreferably mammals selected from the group of humans, non-humanprimates, canines, felines, murines, bovines, equines, porcines, andlagomorphs.

Preferred biological samples are selected from the group consisting ofexcised tissue (e.g., tissue biopsy), whole blood, serum, plasma, buccalscrape, saliva, cerebrospinal fluid, and urine.

In preferred embodiments, the difference between the increased level ofCYP24 in the biological sample and the level of CYP24 in said controlsample is a statistically significant difference (e.g. the increasedlevel of CYP24 in the biological sample is at least about 2-foldgreater, more preferably at least 4-fold greater than the level of CYP24in the control sample).

This invention also provides methods of treating cancer in an animal.The methods involve performing the assays as described herein (e.g.providing a biological sample from said animal; detecting the level ofCYP24 within said biological sample; and comparing said level of CYP24with a level of CYP24 in a control sample from a normal, cancer-freetissue) and selecting and performing a cancer therapy in those animalshaving an increased level of CYP24 compared to the level of CYP24 insaid control sample. In preferred embodiments, the cancer therapy isselected from the group consisting of chemotherapy, radiation therapy,surgery, antihormone therapy, and immunotherapy. In some preferredembodiments, the cancer therapy is an adjuvant cancer therapy.

This invention also provides methods of screening a test agent for theability to inhibit proliferation of a CYP24-expressing cell. The methodsinvolve contacting the CYP24-expressing cell with said test agent; anddetecting the level of CYP24 activity, where a decreased level of CYP24activity as compared to the level of CYP24 activity in a cell notcontacted with the agent indicates that the agent inhibits proliferationof said cell. In a preferred embodiment, the cell is contacted withvitamin D. The detection of CYP24 level can be as described herein. Insome embodiments the CYP24-expressing cell is a tumor cell. In someembodiments, the CYP24-expressing cell is a hyperproliferative cell. Inparticularly preferred embodiments, the difference between saiddecreased level of CYP24 activity and the level of CYP24 activity in acell not contacted with said agent is a statistically significantdifference (e.g. at least 2-fold lower, more preferably at least 4-foldlower in the cell contacted with the test agent).

This invention additionally provides methods of decreasing theproliferation of a cell with an elevated level of CYP24. The methodsinvolve reducing the level of CYP24 activity in the cell using aninhibitor of CYP24. The methods can further involve contacting the cellwith vitamin D. The cell can be a tumor cell (e.g., breast cancer cell,prostate cancer cell, colorectal cancer cell, leukemia cell, lymphoma,lung cancer cell, brain cancer cell, pancreatic cancer cell, coloncancer cell, and ovarian cancer cell). The cell can be ahyperproliferative cell. The cell can also be a metastatic cell.Preferred inhibitors include antisense oligonucleotides, ribozymes,repressors of CYP24 gene expression, competitive inhibitors of25-hydroxyvitamin D3 24-hydroxylase activity, and non-competitiveinhibitors of 25-hydroxyvitamin D3 24-hydroxylase activity.

DEFINITIONS

To facilitate understanding the invention, a number of terms are definedbelow.

A “CYP24 gene” is a DNA sequence that encodes a 25-hydroxyvitamin D324-hydroxylase enzyme (see, e.g. GenBank Accession Numbers U60669 578775and X59506). The term gene can refer to a mutated copy of the gene, or afragment of the gene.

The term “VDR” refers to a vitamin D receptor.

The terms “hybridizing specifically to” and “specific hybridization” and“selectively hybridize to,” as used herein refer to the binding,duplexing, or hybridizing of a nucleic acid molecule preferentially to aparticular nucleotide sequence under stringent conditions. The term“stringent conditions” refers to conditions under which a probe willhybridize preferentially to its target subsequence, and to a lesserextent to, or not at all to, other sequences. A “stringenthybridization” and “stringent hybridization wash conditions” in thecontext of nucleic acid hybridization (e.g., as in array, Southern orNorthern hybridizations) are sequence dependent, and are different underdifferent environmental parameters. An extensive guide to thehybridization of nucleic acids is found in, e.g., Tijssen (1993)Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes part I, chapt 2,“Overview of principles of hybridization and the strategy of nucleicacid probe assays,” Elsevier, NY (“Tijssen”). Generally, highlystringent hybridization and wash conditions are selected to be about 5°C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Very stringentconditions are selected to be equal to the T_(m) for a particular probe.An example of stringent hybridization conditions for hybridization ofcomplementary nucleic acids which have more than 100 complementaryresidues on an array or on a filter in a Southern or northern blot is42° C. using standard hybridization solutions (see, e.g., Sambrook(1989) Molecular Cloning: A Laboratory Manual (2nd ed.) Vol. 1-3, ColdSpring Harbor Laboratory, Cold Spring Harbor Press, NY, and detaileddiscussion, below), with the hybridization being carried out overnight.An example of highly stringent wash conditions is 0.15 M NaCl at 72° C.for about 15 minutes. An example of stringent wash conditions is a0.2×SSC wash at 65° C. for 15 minutes (see, e.g., Sambrook supra.) for adescription of SSC buffer). A typical stringent wash for an arrayhybridization is 50% formamide, 2×SSC at 50° C. to 50° C. Often, a highstringency wash is preceded by a low stringency wash to removebackground probe signal. An example medium stringency wash for a duplexof, e.g., more than 100 nucleotides, is 1×SSC at 45° C. for 15 minutes.An example of a low stringency wash for a duplex of, e.g., more than 100nucleotides, is 4× to 6×SSC at 40° C. for 15 minutes.

The term “labeled with a detectable composition”, as used herein, refersto a nucleic acid attached to a detectable composition, i.e., a label.The detection can be by, e.g., spectroscopic, photochemical,biochemical, immunochemical, physical or chemical means. For example,useful labels include ³²P, ³⁵S, ³H, ¹⁴C, ¹²⁵I, ¹³¹I; fluorescent dyes(e.g., FITC, rhodamine, lanthanide phosphors, Texas red), electron-densereagents (e.g. gold), enzymes, e.g., as commonly used in an ELISA (e.g.,horseradish peroxidase, beta-galactosidase, luciferase, alkalinephosphatase), colorimetric labels (e.g. colloidal gold), magnetic labels(e.g. Dynabeads™), biotin, dioxigenin, or haptens and proteins for whichantisera or monoclonal antibodies are available. The label can bedirectly incorporated into the nucleic acid, peptide or other targetcompound to be detected, or it can be attached to a probe or antibodythat hybridizes or binds to the target. A peptide can be made detectableby incorporating predetermined polypeptide epitopes recognized by asecondary reporter (e.g., leucine zipper pair sequences, binding sitesfor secondary antibodies, transcriptional activator polypeptide, metalbinding domains, epitope tags). Label can be attached by spacer arms ofvarious lengths to reduce potential steric hindrance or impact on otheruseful or desired properties (see, e.g., Mansfield (1995) Mol CellProbes 9: 145-156). It will be appreciated that combinations of labelscan also be used. Thus, for example, in some embodiments, differentnucleic acids may be labeled with distinguishable (e.g. differentlycolored) labels.

The term “nucleic acid” as used herein refers to a deoxyribonucleotideor ribonucleotide in either single- or double-stranded form. The termencompasses nucleic acids, i.e., oligonucleotides, containing knownanalogues of natural nucleotides which have similar or improved bindingproperties, for the purposes desired, as the reference nucleic acid. Theterm also includes nucleic acids which are metabolized in a mannersimilar to naturally occurring nucleotides or at rates that are improvedthereover for the purposes desired. The term also encompassesnucleic-acid-like structures with synthetic backbones. DNA backboneanalogues provided by the invention include phosphodiester,phosphorothioate, phosphorodithioate, methylphosphonate,phosphoramidate, alkyl phosphotriester, sulfamate, 3′-thioacetal,methylene(methylimino), 3′-N-carbamate, morpholino carbamate, andpeptide nucleic acids (PNAs); see Oligonucleotides and Analogues, aPractical Approach, edited by F. Eckstein, IRL Press at OxfordUniversity Press (1991); Antisense Strategies, Annals of the New YorkAcademy of Sciences, Volume 600, Eds. Baserga and Denhardt (NYAS 1992);Milligan (1993) J. Med. Chem. 36:1923-1937; Antisense Research andApplications (1993, CRC Press). PNAs contain non-ionic backbones, suchas N-(2-aminoethyl) glycine units. Phosphorothioate linkages aredescribed in WO 97/03211; WO 96/39154; Mata (1997) Toxicol. Appl.Pharmacol. 144:189-197. Other synthetic backbones encompasses by theterm include methyl-phosphonate linkages or alternatingmethylphosphonate and phosphodiester linkages (Strauss-Soukup (1997)Biochemistry 36: 8692-8698), and benzylphosphonate linkages (Samstag(1996) Antisense Nucleic Acid Drug Dev 6: 153-156). The term nucleicacid is used interchangeably with gene, cDNA, mRNA, oligonucleotideprimer, probe and amplification product.

The term a “nucleic acid array” as used herein is a plurality of targetelements, each target element comprising one or more nucleic acidmolecules (probes) immobilized on one or more solid surfaces to whichsample nucleic acids can be hybridized. The nucleic acids of a targetelement can contain sequence(s) from specific genes or clones, e.g. fromCYP24. Other target elements will contain, for instance, referencesequences. Target elements of various dimensions can be used in thearrays of the invention. Generally, smaller, target elements arepreferred. Typically, a target element will be less than about 1 cm indiameter. Generally element sizes are from 1 μm to about 3 mm,preferably between about 5 μm and about 1 mm. The target elements of thearrays may be arranged on the solid surface at different densities. Thetarget element densities will depend upon a number of factors, such asthe nature of the label, the solid support, and the like. One of skillwill recognize that each target element may comprise a mixture ofnucleic acids of different lengths and sequences. Thus, for example, atarget element may contain more than one copy of a cloned piece of DNA,and each copy may be broken into fragments of different lengths. Thelength and complexity of the nucleic acid fixed onto the target elementis not critical to the invention. One of skill can adjust these factorsto provide optimum hybridization and signal production for a givenhybridization procedure, and to provide the required resolution amongdifferent genes or genomic locations. In various embodiments, targetelement sequences will have a complexity between about 1 kb and about 1Mb, between about 10 kb to about 500 kb, between about 200 to about 500kb, and from about 50 kb to about 150 kb.

The term “probe” or a “nucleic acid probe”, as used herein, is definedto be a collection of one or more nucleic acid fragments whosehybridization to a sample can be detected. The probe may be unlabeled orlabeled as described below so that its binding to the target or samplecan be detected. Particularly in the case of arrays, either probe ortarget nucleic acids may be affixed to the array. Whether the arraycomprises “probe” or “target” nucleic acids will be evident from thecontext. Similarly, depending on context, either the probe, the target,or both can be labeled. The probe is produced from a source of nucleicacids from one or more particular (preselected) portions of the genome,e.g., one or more clones, an isolated whole chromosome or chromosomefragment, or a collection of polymerase chain reaction (PCR)amplification products. The probes of the present invention are producedfrom nucleic acids found in the regions described herein. The probe orgenomic nucleic acid sample may be processed in some manner, e.g., byblocking or removal of repetitive nucleic acids or enrichment withunique nucleic acids. The word “sample” may be used herein to refer notonly to detected nucleic acids, but to the detectable nucleic acids inthe form in which they are applied to the target, e.g., with theblocking nucleic acids, etc. The blocking nucleic acid may also bereferred to separately. What “probe” refers to specifically is clearfrom the context in which the word is used. The probe may also beisolated nucleic acids immobilized on a solid surface (e.g.,nitrocellulose, glass, quartz, fused silica slides), as in an array. Insome embodiments, the probe may be a member of an array of nucleic acidsas described, for instance, in WO 96/17958. Techniques capable ofproducing high density arrays can also be used for this purpose (see,e.g., Fodor (1991) Science 767-773; Johnston (1998) Curr. Biol. 8:R171-R174; Schummer (1997) Biotechniques 23: 1087-1092; Kern (1997)Biotechniques 23: 120-124; U.S. Pat. No. 5,143,854). One of skill willrecognize that the precise sequence of the particular probes describedherein can be modified to a certain degree to produce probes that are“substantially identical” to the disclosed probes, but retain theability to specifically bind to (i.e., hybridize specifically to) thesame targets or samples as the probe from which they were derived (seediscussion above). Such modifications are specifically covered byreference to the individual probes described herein.

The term “sample of human nucleic acid” as used herein refers to asample comprising human DNA or RNA in a form suitable for detection byhybridization or amplification. The nucleic acid may be isolated, clonedor amplified; it may be, e.g., genomic DNA, mRNA, or cDNA from aparticular chromosome, or selected sequences (e.g. particular promoters,genes, amplification or restriction fragments, cDNA, etc.) withinparticular amplicons or deletions disclosed here. The nucleic acidsample may be extracted from particular cells or tissues. The cell ortissue sample from which the nucleic acid sample is prepared istypically taken from a patient suspected of having cancer associatedwith the amplicon amplification or deletion or translocation beingdetected. Methods of isolating cell and tissue samples are well known tothose of skill in the art and include, but are not limited to,aspirations, tissue sections, needle biopsies, and the like. Frequentlythe sample will be a “clinical sample” which is a sample derived from apatient, including sections of tissues such as frozen sections orparaffin sections taken for histological purposes. The sample can alsobe derived from supernatants (of cells) or the cells themselves fromcell cultures, cells from tissue culture and other media in which it maybe desirable to detect chromosomal abnormalities or determine ampliconcopy number. In some cases, the nucleic acids may be amplified usingstandard techniques such as PCR, prior to the hybridization. The samplemay be isolated nucleic acids immobilized on a solid. In one embodiment,the sample may be prepared such that individual nucleic acids remainsubstantially intact and typically comprises interphase nuclei preparedaccording to standard techniques.

The phrase “detecting a cancer” refers to the ascertainment of thepresence or absence of cancer in an animal. “Detecting a cancer” canalso refer to obtaining indirect evidence regarding the likelihood ofthe presence of cancerous cells in the animal or to the likelihood orpredilection to development of a cancer. Detecting a cancer can beaccomplished using the methods of this invention alone, or incombination with other methods or in light of other informationregarding the state of health of the animal.

A “cancer” in an animal refers to the presence of cells possessingcharacteristics typical of cancer-causing cells, such as uncontrolledproliferation, immortality, metastatic potential, rapid growth andproliferation rate, and certain characteristic morphological features.Often, cancer cells will be in the form of a tumor, but such cells mayexist alone within an animal, or may be a non-tumorigenic cancer cell,such as a leukemia cell. Cancers include, but are not limited to breastcancer, lung cancer, bronchus cancer, colorectal cancer, prostatecancer, pancreas cancer, stomach cancer, ovarian cancer, urinary bladdercancer, brain or central nervous system cancer, peripheral nervoussystem cancer, esophageal cancer, cervical cancer, a melanoma, uterineor endometrial cancer, cancer of the oral cavity or pharynx, livercancer, kidney cancer, testis cancer, biliary tract cancer, small bowelor appendix cancer, salivary gland cancer, thyroid gland cancer, adrenalgland cancer, osteosarcoma, and a chondrosarcoma.

An “animal” refers to a member of the kingdom Animalia, characterized bymulticellularity, the possession of a nervous system, voluntarymovement, internal digestion, etc. An “animal” can be a human or othermammal. Preferred animals include humans, non-human primates, and othermammals. Thus, it will be recognized that the methods of this inventioncontemplate veterinary applications as well as medical applicationsdirected to humans.

“Providing a biological sample” means to obtain a biological sample foruse in the methods described in this invention. Most often, this will bedone by removing a sample of cells from an animal, but can also beaccomplished by using previously isolated cells (e.g. isolated byanother person), or by performing the methods of the invention in vivo.

A “biological sample” refers to a cell or population of cells or aquantity of tissue or fluid from an animal. Most often, the sample hasbeen removed from an animal, but the term “biological sample” can alsorefer to cells or tissue analyzed in vivo, i.e. without removal from theanimal. Often, a “biological sample” will contain cells from the animal,but the term can also refer to non-cellular biological material, such asnon-cellular fractions of blood, saliva, or urine, that can be used tomeasure CYP24 levels. Preferred biological samples include tissuebiopsies, scrapes (e.g. buccal scrapes), whole blood, plasma, serum,urine, saliva, cell culture, or cerebrospinal fluid.

“Detecting a level of CYP24” refers to determining the number of CYP24genes or the expression level of a gene or genes encoding25-hydroxyvitamin D3 24-hydroxylase enzyme. The copy number of a genecan be measured in multiple ways known to those of skill in the art,including, but not limited to, Comparative Genomic Hybridization (CGH)and quantitative DNA amplification (e.g. quantitative PCR). Geneexpression can be monitored in a variety of ways, including by detectingmRNA levels, protein levels, or protein activity, any of which can bemeasured using standard techniques. Detection can involve quantificationof the level of CYP24 (e.g. genomic DNA, cDNA, mRNA, protein, or enzymeactivity), or, alternatively, can be a qualitative assessment of thelevel of CYP24, in particular in comparison with a control level. Thetype of level being detected will be clear from the context. BecauseCYP24 activity is tightly linked to VDR activity, measurement of geneexpression is preferably done in combination with a measurement of VDRactivity.

To “compare” levels of CYP24 means to detect CYP24 levels in two samplesand to determine whether the levels are equal or if one or the other isgreater. A comparison can be done between quantified levels, allowingstatistical comparison between the two values, or in the absence ofquantification, for example using qualitative methods of detection suchas visual assessment by a human.

A “control sample” refers to a sample of biological materialrepresentative of healthy, cancer-free animals, and/or cells or tissues.The level of CYP24 in a control sample is desirably typical of thegeneral population of normal, cancer-free animals or of a particularindividual at a particular time (e.g. before, during or after atreatment regimen), or in a particular tissue. This sample can beremoved from an animal expressly for use in the methods described inthis invention, or can be any biological material representative ofnormal, cancer-free animals, including cancer-free biological materialtaken from an animal with cancer elsewhere in its body. A control samplecan also refer to an established level of CYP24, representative of thecancer-free population, that has been previously established based onmeasurements from normal, cancer-free animals.

An “increased level of CYP24” means a level of CYP24, that, incomparison with a control level of CYP24, is detectably higher. Themethod of comparison can be statistical, using quantified values for thelevel of CYP24, or can be compared using non-statistical means, such asby visual assessment by a human.

The “copy number of CYP24 genes” refers to the number of DNA sequencesin a cell encoding a 25-hydroxyvitamin D3 24-hydroxylase enzyme.Generally, for a given gene, an animal has two copies of each gene. Thecopy number can be increased, however, by gene amplification orduplication, or reduced by deletion.

When a level of CYP24 mRNA, protein, enzyme activity, or copy number is“measured,” it is assessed using qualitative or quantitative methods.Preferably, the level is determined using quantitative means, allowingthe statistical comparison of values obtained from biological samplesand control values. The level can also be determined using qualitativemethods, such as the visual analysis and comparison by a human ofmultiple visibly labeled samples, e.g. fluorescently labeled samplesdetected using a fluorescent microscope or other optical detector (e.g.image analysis system, etc.). When a level of CYP24 mRNA, protein, orenzyme activity is measured the measurement preferably includes ameasurement of VDR activity, and/or a measure of CYP24 activity in anormal tissue or cell (e.g. from the same animal or from a different“control” animal).

“25-hydroxyvitamin D3 24-hydroxylase enzyme activity” means thecatalysis of the 24-hydroxylation of 25-hydroxyvitamin D3, 1,alpha-25dihydroxyvitamin D3, or other analogous substrates (see, e.g., Stryer(1988) Biochemistry, 3^(rd) Ed., W.H. Freeman and Co.; Jehan et al.,(1998) Biochim Biophys Acta 1395:259-265; Seo and Norman (1997) J BoneMiner Res 12:598-606).

“Tissue biopsy” refers to the removal of a biological sample fordiagnostic analysis. In a patient with cancer, tissue may be removedfrom a tumor, allowing the analysis of cells within the tumor.

When a quantified level of CYP24 falls outside of a given confidenceinterval for a normal level of CYP24, the difference between the twolevels is said to be “statistically significant.” If a test value fallsoutside of a given confidence interval for a normal level of CYP24, itis possible to calculate the probability that the test value is trulyabnormal and does not just represent a normal deviation from theaverage. In the methods of this invention, a difference between a testsample and a control can be termed “statistically significant” when theprobability of the test sample being abnormal can be any of a number ofvalues, including 0.15, 0.1, 0.05, and 0.01. Numerous sources teach howto assess statistical significance, such as Freund, J. E. (1988) Modernelementary statistics, Prentice-Hall.

The “survival expectancy” of an animal refers to a prognostic estimateof the outcome of a disease or condition. A “survival expectancy” canrefer to a prediction regarding the severity, duration, or progress of adisease, condition, or any symptom thereof. “Survival expectancy” canalso refer to the length of time an animal is expected to survive, or tothe probability of the animal surviving until a certain time.

A “method of treating cancer” refers to a procedure or course of actionthat is designed to reduce or eliminate the number of cancer cells in ananimal, or to alleviate the symptoms of a cancer. “A method of treatingcancer” does not necessarily mean that the cancer cells will in fact beeliminated, that the number of cells will in fact be reduced, or thatthe symptoms of a cancer will in fact be alleviated. Often, a method oftreating cancer will be performed even with a low likelihood of success,but which, given the medical history and estimated survival expectancyof an animal, is deemed an overall beneficial course of action.

“Reducing the level of CYP24 activity” refers to inhibiting the25-hydroxyvitamin D3 24-hydroxylase enzyme activity in the cell, orlowering the copy number of CYP24 genes, or decreasing the level ofCYP24 mRNA or protein in the cell (e.g., at a given VDR activity level).Preferably, the level of CYP24 activity is lowered to the level typicalof a normal, cancer-free cell, but the level may be reduced to any levelthat is sufficient to decrease the proliferation of the cell, includingto levels below those typical of normal cells.

“Contacting” a cell with vitamin D is to ensure that the cell is in thepresence of vitamin D. In the case of a cell that is not naturally incontact with vitamin D, vitamin D is added to the cell, in vivo or invitro. “Vitamin D” refers to any of the family of vitamin D molecules,including but not limited to vitamin D1, vitamin D2, and vitamin D3. Italso refers to structural and functional homologs of these molecules,e.g. those that are substrates for the CYP24 enzyme, as well asmetabolic products of vitamin D.

A “tumor cell” is a cancer cell that is part of a tumor, has beenisolated from a tumor, or which is capable of forming a tumor. Tumorcells can exist in vivo or in vitro.

A “hyperproliferative cell” is a cell with an abnormally high rate ofproliferation, or a cell that proliferates to an abnormally greatextent, i.e. gives rise to a population of cells that increases innumber over time. “Hyperproliferative cells” can exist in vitro or invivo.

An “inhibitor of CYP24 activity” is a molecule that acts to reduce CYP24activity, as defined above. Such inhibitors can include antisensemolecules or ribozymes, repressors of CYP24 gene transcription, orcompetitive or non-competitive molecular inhibitors of the25-hydroxyvitamin D3 24-hydroxylase enzyme.

The phrase “repressor of CYP24 transcription” refers to a molecule thatcan prevent the production of CYP24 mRNA from a CYP24 gene. Preferably,the molecule binds directly or indirectly to a regulatory element of theCYP24 gene, thereby preventing the transcription of the CYP24 gene.

A “competitive inhibitor of 25-hydroxyvitamin D3 24-hydroxylase” means amolecule that can bind directly or indirectly to a 25-hydroxyvitamin D324-hydroxylase enzyme or to its substrate, thereby preventing thebinding of the enzyme to its substrate and preventing the24-hydroxylation of the substrate, in vitro or in vivo.

The phrase “non-competitive inhibitor of 25-hydroxyvitamin D324-hydroxylase” refers to a molecule that prevents the 24-hydroxylationof a 25-hydroxyvitamin D3 24-hydroxylase enzyme substrate but which doesnot prevent the binding of the enzyme to the substrate.

“Screening” for an inhibitor of cell proliferation or of CYP24 activitymeans to systematically examine the ability of a population of moleculesto inhibit cell proliferation or CYP24 activity. Screening can be donein vitro or in vivo. The inhibitory activity of the screened moleculescan be assessed directly, e.g. by examining CYP24 enzyme activity usingstandard assays, or indirectly, e.g. by monitoring a cellularconsequence of CYP24 enzyme activity, such as cell proliferation.

A “CYP24-expressing cell” is a cell that produces any amount of25-hydroxyvitamin D3 24-hydroxylase protein. Generally, the amount ofenzyme produced by the cell will be detectable using standardtechniques.

A “test agent” is any molecule or non-molecular entity that is tested ina screen. The molecule may be randomly selected for inclusion in thescreen, or may be included because of an a priori expectation that themolecule will give a positive result in the screen. The molecule may bedirectly introduced into a cell or a biochemical assay for the purposesof the screen, or it may comprise nucleic acids that encode apolypeptide or RNA that is desirably tested in the screen. Moleculesintroduced directly into an assay system can include any known chemicalor biochemical molecule, including peptides, nucleic acids,carbohydrates, lipids, or any other organic or inorganic molecule. A“test agent” can also refer to non-molecular entities such aselectromagnetic radiation or heat.

The “proliferation” of a cell refers to the rate at which the cell orpopulation of cells grows and divides, or to the extent to which thecell or population of cells grows, divides, or increases in number. The“proliferation” of a cell can reflect multiple factors including therate of cell growth and division and the rate of cell death.

The phrase “decreasing the proliferation of a cell” means to reduce therate or extent of growth or division of a cell or population of cells.Such methods can involve preventing cell division or cell growth, andmay also include cell killing, and can be practiced in vivo or in vitro.

“CYP24-inhibiting activity” is the ability of a molecule to reduce orprevent the production and/or accumulation of 25-hydroxyvitamin D324-hydroxylase enzyme activity in a cell. The molecule can prevent theaccumulation at any step of the pathway from the CYP24 gene to enzymeactivity, e.g. preventing transcription, reducing mRNA levels,preventing translation, or inhibiting the enzyme itself. The reductionor prevention is preferably ascertained by reference to a control at thesame level of VDR activity.

A CYP24 enzyme or CYP24 polypeptide is a protein with 25-hydroxyvitaminD3 24-hydroxylase activity and is most preferably encoded by a CYP24gene.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers.

An amino acid, identified by name herein “e.g., arginine” or “arginineresidue” as used herein refers to natural, synthetic, or version of theamino acids Thus, for example, an arginine can also include arginineanalogs that offer the same or similar functionality as natural argininewith respect to their ability to be incorporated into a polypeptide,effect folding of that polypeptide and effect interactions of thatpolypeptide with other polypeptide(s).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates Comparative Genomic Hybridization (CGH). In the leftpanel, total genomic DNAs are isolated from a “test” and a “reference”cell population, labeled with different fluorochromes, and hybridized tonormal metaphase chromosomes. Cot-1 DNA is used to suppresshybridization of repetitive sequences. The resulting ratio of thefluorescence intensities of the two fluorochromes at a location on achromosome is approximately proportional to the ratio of the copynumbers of the corresponding DNA sequences in the test and referencegenomes. The right panel shows a similar hybridization to an array ofmapped clones. This permits measurement of copy number with resolutiondetermined by the size of the clones and/or the map spacing betweenthem.

FIG. 2 illustrates a high resolution array CGH measurement on the breastcancer tumor, S21. The test:reference ratios for contiguous targetclones from region A at 20q13.2 are plotted with the ratio on eachtarget clone shown as a bar, representing the clone overlaps asdetermined by STS-content mapping. Clone names have been shortened tothe last four digits. The analysis indicates that the overlappingclones, RMC20B4121, RMC20B4087, and RMC20B4195 are the locus of the peakin the copy number profile and that copy number in the region decreasesprecipitously distal to the overlapping clones, RMC20B4087 andRMC20B4195.

FIG. 3 illustrates expression of CYP24, VDR and ZNF217 genes in humanbreast cancer cell lines and tumors with and without induction by1,25-dihydroxyvitamin D₃ evaluated by RT-PCR. (a) Time course ofinduction of CYP24 gene expression in MCF7 breast cancer cells incubatedwith 10⁻⁸M 1,25-dihydroxyvitamin D₃ and vehicle control (ethanol, EtOH).(b) Gene expression in two breast cancer tumors, S21 and S59. (c) Geneexpression in the BT474 cell line. Cells were incubated with 10⁻⁸M1,25-dihydroxyvitamin D₃, as described in (a).

DETAILED DESCRIPTION

This invention pertains to the discovery that that amplification of thevitamin D 24 hydroxylase (CYP24) gene (GenBank Accession Numbers U60669S78775 and X59506) occurs in various cancers (e.g., breast tumors).Vitamin D 24 hydroxylase controls activity of the vitamin D system incells by initiating degradation of the active form of vitamin D3.Without being bound by a particular theory, it is believed thatamplification of CYP24 during tumor evolution provides a means todisrupt vitamin D mediated growth control.

Amplification of chromosome band 20q13.2 in human breast cancer isassociated with poor prognosis and aggressive tumor behavior (Tanner etal., (1995) Clin. Cancer Res. 1: 1455-1461; Courjal et al. (1996) Br. J.Cancer, 74: 1984-1989), suggesting that overexpression of genes mappingto this region is likely to contribute to the development of breastcancer. Using a new high resolution form of comparative genomichybridization, array CGH (Pinkel et al. (1998) Nature Genetics, 20:207-211), we mapped DNA copy number profiles across the region ofrecurrent amplification at 20q13.2.

This analysis focused attention on the gene CYP24, because it mapped tothe narrow genomic interval that is most highly amplified in the mostinformative tumors and because of existing knowledge of CYP24 function.CYP24 encodes vitamin D 24 hydroxylase, an enzyme that catalyzesdegradation of the active form of vitamin D, 1,25-dihydroxy-D3 (forreviews, see Walters (1992) Endocrine Reviews 13: 719-764; Jones et al.(1998) Amer. Physiol. Soc. 78: 1193-1231). Vitamin D is a secosteroidhormone that plays a major role in the regulation of calcium and bonemetabolism. However, vitamin D receptors (VDR) have also been found inmany other so-called “non-classical” tissues not involved in mineralmetabolism, including the breast (Berger et al. (1987) Cancer Res. 47:6793-6799; Buras et al. (1994) Breast Cancer Res. and Treatment 31:191-202), indicating a role for vitamin D in these tissues also. Levelsof 1,25-dihydroxy-D3 and ligand bound receptor appear to be very tightlycontrolled in cells by a feedback mechanism. Binding of the hormone tothe VDR results in activation of CYP24 transcription to initiatedegradation of 1,25-dihydroxy-D3 and inhibition of CYP1, the enzymerequired for synthesis of 1,25-dihydroxy-D3. In fact, transcription ofCYP24 is so closely coupled to VDR levels and activity that activationof transcription from a CYP24 promoter-reporter construct is used as anassay for VDR activity (Arbour et al. (1998) Anal. Biochem. 255:148-154). Thus, without being bound to this theory, we believe the roleof CYP24 in cells is to limit the biological activity of the vitamin Dsystem.

In the “non-classical” tissues such as breast, vitamin D promotes growthinhibition by directing cells towards differentiation and cessation ofproliferation. Breast cancer cells respond to the antiproliferativeeffects of vitamin D both in vivo and in vitro (Eisman et al. (1989)Cancer Res. 47: 21-25). Breast cancer cell lines generally arrest in theG0/G1 stage of the cell cycle in response to vitamin D, and the MCF-7breast cancer cell line can be induced to enter apoptosis (Elstner etal. (1995) Cancer Res. 55: 2822-2830; Love-Schimenti et al. (1996)Cancer Res. 56: 2789-2794; Simboli-Campbell et al. (1997) Breast CancerRes. and Treatment, 42: 31-41). Administration of vitamin D to rodentsreduces progression of tumor xenographs (Eisman et al. (1989) CancerRes. 47: 21-25; Colston et al. (1989) Lancet, 188-191).

These growth modulatory properties of vitamin D support the presentbelief that disruption of the vitamin D system is likely to contributeto neoplasia. This suggestion is further supported by the observationthat patients with receptor negative tumors have a poorer prognosis andby epidemiological studies that have established that exposure tosunlight and risk of breast and colon cancer (Gorham et al. (1989) Can.J. Public Health 80: 96-100; Gorham et al. (1990) Int. J. Epidemiol. 19:820-824; Garland et al. (1990) Preventive Medicine 19: 614-622) areinversely correlated.

Thus, the present hypothesized oncogenic role of CYP24 derives from itsfunction to reduce levels of 1,25-dihydroxyvitamin-D3 and so modulatethe biological effects of ligand bound VDR. This hypothesis is supportedby the observation that the antiproliferative activity of vitamin D invitro is enhanced in the presence of hydroxylase inhibitors (Reinhardtand Horst (1989) Arch. Biochem. Biophys. 272: 459-465; Zhao et al.(1996) J. Steroid. Biochem. Mol. Biol. 57: 197-202). Thus, without beingbound by a theory, the present invention is predicated, in part, on therecognition that amplification of CYP24 abrogates vitamin D mediatedgrowth control by up-regulation of vitamin D degradation in cells, sinceligand bound VDR will bind to and initiate transcription from anincreased number of CYP24 gene copies.

In view of these discoveries, CYP24 provides a good marker for a cancerand/or for the likelihood of (predilection to) development of a cancer.Thus, in one embodiment, this invention provides methods of detectingthe presence of, or a predisposition to, cancer in an animal. Themethods involve (i) providing a biological sample from an animal (e.g. ahuman patient); (ii) detecting the level of CYP24 within the biologicalsample; and (iii) comparing the level of CYP24 with a level of CYP24 ina control sample taken from a normal, cancer-free animal where anincreased level of CYP24 in the biological sample compared to the levelof CYP24 in the control sample indicates the presence of said cancer insaid animal. Where the CYP24 transcript, translated polypeptide, orenzymatic activity is assayed, the methods preferably include ameasurement of VDR activity and the comparison between sample andcontrol is made at the same VDR level or corrections are made reflectingdifferences in VDR level.

Similarly, the detection of CYP24 level can also be used to estimate thesurvival expectancy of an animal with cancer. Because CYP24 level can beused to assay survival expectancy (e.g. likelihood of progression orrecurrence of the disease), an assay of CYP24 level provides a usefulcomponent of a cancer therapy. Thus, in one preferred method of treatingcancer, CYP24 level is assayed and, where it is high relative to theappropriate control or population standard, one or more adjuvanttherapies (e.g. radiation therapy, resurgery, chemotherapy, etc.) areselected for the cancer treatment regimen.

Having identified elevated CYP24 levels as indicative of a cancer or apredisposition to cancer, CYP24 level provides a useful target/markerfor evaluating potential prophylaxis and/or therapeutics. Thus, forexample, the level of CYP24 activity (at a given level of VDR activity)in the presence or absence of one or more putative potentialtherapeutics or prophylactics provides a measure of the potentialactivity of the therapeutic/prophylactic compound, i.e., a lower CYP24activity in the presence of the compound indicates higher potentialactivity of the compound.

In another embodiment this invention provides a method of decreasing theproliferation of a cell (e.g. a cancer cell). The method involvesreducing the level of CYP24 activity in said cell using an inhibitor ofCYP24.

I. Assays of CYP24 Level.

As indicated above, assays of CYP24 copy number or level of activity(e.g., at a particular vitamin D receptor activity) provide a measure ofthe presence or likelihood of (predisposition to) a cancer. The sequenceof CYP24 is known and hence, copy number can be directly measuredaccording to a number of different methods as described below.

With respect to assays based on CYP24 “activity” level (e.g., level oftranscript, level of translated protein, level of protein enzymaticactivity), the close coupling of transcription of CYP24 to vitamin Dreceptor (VDR) level and activity complicates the evaluation of CYP24level. In short, CYP24 expression levels depend on VDR activity as wellas the magnitude of transcription resulting from copy number increases.Thus, particularly in embodiments relying on assays of CYP24 “activity”,evaluation of CYP24 levels preferably involves measurement not only ofCYP24 levels in tumor cells relative to normal tissue, but also the VDRlevels and activities in the tumors and normal tissues. Such assays aredescribed below.

A) Detection of Copy Number

In one embodiment, the presence of, or predilection to cancer, isevaluated simply by a determination of CYP24 copy number. Methods ofevaluating the copy number of a particular gene are well known to thoseof skill in the art.

1) Hybridization-Based Assays

One method for evaluating the copy number of CYP24-encoding nucleic acidin a sample involves a Southern transfer. In a Southern Blot, thegenomic DNA (typically fragmented and separated on an electrophoreticgel) is hybridized to a probe specific for the target region. Comparisonof the intensity of the hybridization signal from the probe for thetarget region with control probe signal from analysis of normal genomicDNA (e.g., a non-amplified portion of the same or related cell, tissue,organ, etc.) provides an estimate of the relative copy number of thetarget nucleic acid.

An alternative means for determining the copy number of CYP24 is in situhybridization. In situ hybridization assays are well known (e.g.,Angerer (1987) Meth. Enzymol 152: 649). Generally, in situ hybridizationcomprises the following major steps: (1) fixation of tissue orbiological structure to be analyzed; (2) prehybridization treatment ofthe biological structure to increase accessibility of target DNA, and toreduce nonspecific binding; (3) hybridization of the mixture of nucleicacids to the nucleic acid in the biological structure or tissue; (4)post-hybridization washes to remove nucleic acid fragments not bound inthe hybridization and (5) detection of the hybridized nucleic acidfragments. The reagent used in each of these steps and the conditionsfor use vary depending on the particular application.

Preferred hybridization-based assays include, but are not limited to,traditional “direct probe” methods such as Southern blots or in situhybridization (e.g., FISH), and “comparative probe” methods such ascomparative genomic hybridization (CGH). The methods can be used in awide variety of formats including, but not limited to substrate- (e.g.membrane or glass) bound methods or array-based approaches as describedbelow.

In a typical in situ hybridization assay, cells are fixed to a solidsupport, typically a glass slide. If a nucleic acid is to be probed, thecells are typically denatured with heat or alkali. The cells are thencontacted with a hybridization solution at a moderate temperature topermit annealing of labeled probes specific to the nucleic acid sequenceencoding the protein. The targets (e.g., cells) are then typicallywashed at a predetermined stringency or at an increasing stringencyuntil an appropriate signal to noise ratio is obtained.

The probes are typically labeled, e.g., with radioisotopes orfluorescent reporters. Preferred probes are sufficiently long so as tospecifically hybridize with the target nucleic acid(s) under stringentconditions. The preferred size range is from about 200 bp to about 1000bases.

In some applications it is necessary to block the hybridization capacityof repetitive sequences. Thus, in some embodiments, tRNA, human genomicDNA, or Cot-1 DNA is used to block non-specific hybridization.

In comparative genomic hybridization methods a first collection of(sample) nucleic acids (e.g. from a possible tumor) is labeled with afirst label, while a second collection of (control) nucleic acids (e.g.from a healthy cell/tissue) is labeled with a second label. The ratio ofhybridization of the nucleic acids is determined by the ratio of the two(first and second) labels binding to each fiber in the array. Wherethere are chromosomal deletions or multiplications, differences in theratio of the signals from the two labels will be detected and the ratiowill provide a measure of the CYP24 copy number.

Hybridization protocols suitable for use with the methods of theinvention are described, e.g., in Albertson (1984) EMBO J. 3: 1227-1234;Pinkel (1988) Proc. Natl. Acad. Sci. USA 85: 9138-9142; EPO Pub. No.430,402; Methods in Molecular Biology, Vol. 33: In Situ HybridizationProtocols, Choo, ed., Humana Press, Totowa, N.J. (1994), etc. In oneparticularly preferred embodiment, the hybridization protocol of Pinkelet al. (1998) Nature Genetics 20: 207-211, or of Kallioniemi (1992)Proc. Natl Acad Sci USA 89:5321-5325 (1992) is used.

2) Amplification-Based Assays.

In still another embodiment, amplification-based assays can be used tomeasure copy number. In such amplification-based assays, the CYP24nucleic acid sequences act as a template in an amplification reaction(e.g. Polymerase Chain Reaction (PCR). In a quantitative amplification,the amount of amplification product will be proportional to the amountof template in the original sample. Comparison to appropriate (e.g.healthy tissue) controls provides a measure of the copy number of CYP24.

Methods of “quantitative” amplification are well known to those of skillin the art. For example, quantitative PCR involves simultaneouslyco-amplifying a known quantity of a control sequence using the sameprimers. This provides an internal standard that may be used tocalibrate the PCR reaction. Detailed protocols for quantitative PCR areprovided in Innis et al. (1990) PCR Protocols, A Guide to Methods andApplications, Academic Press, Inc. N.Y.). The known nucleic acidsequence for CYP24 (see, GenBank Accession Numbers U60669 S78775 andX59506) is sufficient to enable one of skill to routinely select primersto amplify any portion of the gene.

Other suitable amplification methods include, but are not limited toligase chain reaction (LCR) (see Wu and Wallace (1989) Genomics 4: 560,Landegren et al. (1988) Science 241: 1077, and Barringer et al. (1990)Gene 89: 117, transcription amplification (Kwoh et al. (1989) Proc.Natl. Acad. Sci. USA 86: 1173), self-sustained sequence replication(Guatelli et al. (1990) Proc. Nat. Acad. Sci. USA 87: 1874), dot PCR,and linker adapter PCR, etc.

B) Detection of Gene Expression

As indicated above, CYP24 level can also be assayed as a marker for apredilection to cancer. However, because of the close coupling oftranscription of CYP24 to vitamin D receptor (VDR) level measures ofCYP24 “activity” are preferably coupled with measures of VDR activityfor use in the assays of this invention. Thus, an elevation of CYP24activity, compared to a control at the same level of VDR activity,provides an indication of the presence and/or predilection to a cancer.

In preferred embodiments, CYP24 activity is characterized by a measureof CYP24 gene transcript (e.g. mRNA), by a measure of the quantity oftranslated protein, or by a measure of CYP24 enzymatic activity(25-hydroxyvitamin D3 24-hydroxylase enzyme activity).

1) Detection of Gene Transcript.

a) Direct Hybridization Based Assays.

Methods of detecting and/or quantifying the CYP24 gene transcript (CYP24mRNA or cDNA made therefrom) using nucleic acid hybridization techniquesare known to those of skill in the art (see Sambrook et al. supra). Forexample, one method for evaluating the presence, absence, or quantity ofCYP24 cDNA involves a Southern transfer as described above. Briefly, theCYP24 mRNA is isolated (e.g. using an acid guanidinium-phenol-chloroformextraction method, Sambrook et al. supra.) and reverse transcribed toproduce cDNA. The cDNA is then optionally digested and run on a gels inbuffer and transferred to membranes. Hybridization is then carried outusing the nucleic acid probes specific for the target CYP24 cDNA.

The probes can be full length or less than the full length of thenucleic acid sequence encoding the CYP24 protein. Shorter probes areempirically tested for specificity. Preferably nucleic acid probes are20 bases or longer in length. (See Sambrook et al. for methods ofselecting nucleic acid probe sequences for use in nucleic acidhybridization.) Visualization of the hybridized portions allows thequalitative determination of the presence or absence of CYP24 cDNA.

Similarly, a Northern transfer may be used for the detection of CYP24mRNA directly. In brief, the mRNA is isolated from a given cell sampleusing, for example, an acid guanidinium-phenol-chloroform extractionmethod. The mRNA is then electrophoresed to separate the mRNA speciesand the mRNA is transferred from the gel to a nitrocellulose membrane.As with the Southern blots, labeled probes are used to identify and/orquantify the CYP24 mRNA.

b) Amplification-Based Assays.

In another preferred embodiment, CYP24 transcript (e.g., CYP24 mRNA) canbe measured using amplification (e.g. PCR) based methods as describedabove for directly assessing copy number of CYP24 DNA. In a preferredembodiment, CYP24 transcript level is assessed by using reversetranscription PCR (RT-PCR). As mentioned above, because CYP24 activityis tightly linked to vitamin D receptor (VDR) activity, where genetranscript level is being measured it is preferable to also measure VDRactivity (e.g. transcript level). Then, an increase in CYP24 activityfor a given level of VDR activity indicates a cancer or an increasedpredisposition to cancer. Thus, in preferred amplification-based assays(e.g. RT-PCR) the level of VDR transcript is also assayed.

As indicated above, PCR assay methods are well known to those of skillin the art. Similarly, RT-PCR methods are also well known. Moreover,probes for such an RT-PCR assay are provided below in Table 1 and theassay is illustrated in Example 1 (see, e.g., FIG. 3).

2) Detection of Expressed Protein

The “activity” of CYP24 can also be detected and/or quantified bydetecting or quantifying the expressed CYP24 polypeptide. Thepolypeptide can be detected and quantified by any of a number of meanswell known to those of skill in the art. These may include analyticbiochemical methods such as electrophoresis, capillary electrophoresis,high performance liquid chromatography (HPLC), thin layer chromatography(TLC), hyperdiffusion chromatography, and the like, or variousimmunological methods such as fluid or gel precipitin reactions,immunodiffusion (single or double), immunoelectrophoresis,radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs),immunofluorescent assays, western blotting, and the like.

In one preferred embodiment, the CYP24 polypeptide isdetected/quantified in an electrophoretic protein separation (e.g. a 1-or 2-dimensional electrophoresis). Means of detecting proteins usingelectrophoretic techniques are well known to those of skill in the art(see generally, R. Scopes (1982) Protein Purification, Springer-Verlag,N.Y.; Deutscher, (1990) Methods in Enzymology Vol. 182: Guide to ProteinPurification, Academic Press, Inc., N.Y.).

In another preferred embodiment, Western blot (immunoblot) analysis isused to detect and quantify the presence of CYP24 polypeptide in thesample. This technique generally comprises separating sample proteins bygel electrophoresis on the basis of molecular weight, transferring theseparated proteins to a suitable solid support, (such as anitrocellulose filter, a nylon filter, or derivatized nylon filter), andincubating the sample with the antibodies that specifically bind CYP24polypeptide. The anti-CYP24 polypeptide antibodies specifically bind toCYP24 on the solid support. These antibodies may be directly labeled oralternatively may be subsequently detected using labeled antibodies(e.g., labeled sheep anti-mouse antibodies) that specifically bind tothe anti-CYP24.

In a more preferred embodiment, the CYP24 polypeptide is detected usingan immunoassay. As used herein, an immunoassay is an assay that utilizesan antibody to specifically bind to the analyte (CYP24 polypeptide). Theimmunoassay is thus characterized by detection of specific binding of aCYP24 polypeptide to an anti-CYP24 antibody as opposed to the use ofother physical or chemical properties to isolate, target, and quantifythe analyte.

The CYP24 polypeptide is detected and/or quantified using any of anumber of well recognized immunological binding assays (see, e.g., U.S.Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a reviewof the general immunoassays, see also Asai (1993) Methods in CellBiology Volume 37: Antibodies in Cell Biology, Academic Press, Inc. NewYork; Stites & Terr (1991) Basic and Clinical Immunology 7th Edition.

Immunological binding assays (or immunoassays) typically utilize a“capture agent” to specifically bind to and often immobilize the analyte(in this case CYP24 polypeptide or subsequence). The capture agent is amoiety that specifically binds to the analyte. In a preferredembodiment, the capture agent is an antibody that specifically binds aCYP24 polypeptide. The antibody (anti-CYP24) may be produced by any of anumber of means well known to those of skill in the art.

Immunoassays also often utilize a labeling agent to specifically bind toand label the binding complex formed by the capture agent and theanalyte. The labeling agent may itself be one of the moieties comprisingthe antibody/analyte complex. Thus, the labeling agent may be a labeledCYP24 polypeptide or a labeled anti-CYP24 antibody. Alternatively, thelabeling agent may be a third moiety, such as another antibody, thatspecifically binds to the antibody/CYP24 polypeptide complex.

In one preferred embodiment, the labeling agent is a second human CYP24antibody bearing a label. Alternatively, the second CYP24 antibody maylack a label, but it may, in turn, be bound by a labeled third antibodyspecific to antibodies of the species from which the second antibody isderived. The second can be modified with a detectable moiety, e.g., asbiotin, to which a third labeled molecule can specifically bind, such asenzyme-labeled streptavidin.

Other proteins capable of specifically binding immunoglobulin constantregions, such as protein A or protein G may also be used as the labelagent. These proteins are normal constituents of the cell walls ofstreptococcal bacteria. They exhibit a strong non-immunogenic reactivitywith immunoglobulin constant regions from a variety of species (see,generally Kronval, et al. (1973) J. Immunol., 111: 1401-1406, andAkerstrom (1985) J. Immunol., 135: 2589-2542).

As indicated above, immunoassays for the detection and/or quantificationof CYP24 polypeptide can take a wide variety of formats well known tothose of skill in the art.

Preferred immunoassays for detecting CYP24 polypeptide are eithercompetitive or noncompetitive. Noncompetitive immunoassays are assays inwhich the amount of captured analyte is directly measured. In onepreferred “sandwich” assay, for example, the capture agent (anti-CYP24antibodies) can be bound directly to a solid substrate where they areimmobilized. These immobilized antibodies then capture CYP24 polypeptidepresent in the test sample. The CYP24 thus immobilized is then bound bya labeling agent, such as a second human CYP24 antibody bearing a label.

In competitive assays, the amount of analyte (CYP24 polypeptide) presentin the sample is measured indirectly by measuring the amount of an added(exogenous) analyte (CYP24 polypeptide) displaced (or competed away)from a capture agent (anti CYP24 antibody) by the analyte present in thesample. In one competitive assay, a known amount of, in this case, CYP24polypeptide is added to the sample and the sample is then contacted witha capture agent. The amount of CYP24 polypeptide bound to the antibodyis inversely proportional to the concentration of CYP24 polypeptidepresent in the sample.

In one particularly preferred embodiment, the antibody is immobilized ona solid substrate. The amount of CYP24 polypeptide bound to the antibodymay be determined either by measuring the amount of CYP24 polypeptidepresent in an CYP24 polypeptide/antibody complex, or alternatively bymeasuring the amount of remaining uncomplexed CYP24 polypeptide. Theamount of CYP24 polypeptide may be detected by providing a labeled CYP24polypeptide.

The assays of this invention are scored (as positive or negative orquantity of CYP24 polypeptide) according to standard methods well knownto those of skill in the art. The particular method of scoring willdepend on the assay format and choice of label. For example, a WesternBlot assay can be scored by visualizing the colored product produced bythe enzymatic label. A clearly visible colored band or spot at thecorrect molecular weight is scored as a positive result, while theabsence of a clearly visible spot or band is scored as a negative. Theintensity of the band or spot can provide a quantitative measure ofCYP24.

Antibodies for use in the various immunoassays described herein, can beproduced as described below.

3) Detection of Enzyme Activity.

In another embodiment, CYP24 level (activity) is assayed by measuringthe enzymatic activity of the CYP24 polypeptide (25-hydroxyvitamin D324-hydroxylase enzyme). Methods of assaying the activity of this enzymeare well known to those of skill in the art. Thus, for example, CYP24activity in cell suspensions will be assayed by measuring the metabolismof ³H-labeled 250HD₃ (Amersham). The oxidation products are separated byHPLC and the activity calculated as the sum of the C-24 oxidationproducts (Tomon et al., 1990 Endocrinol., 126: 2868-2875).Alternatively, the CYP24 activity can be determined after incubationwith 25-OH-[26,27-³H]D₃ (NEN #NET349) and measurement of radioactivityreleased as [³H]acetone after periodate cleavage (Beckman and DeLuca(1997) Meth. Enzymol., 282: 200-213).

C) Comparison of CYP24 Levels while Controlling for VDR Activity.

As explained above, the activity level of CYP24 is tightly linked to theactivity level of the vitamin D receptor (VDR). Thus, when assayingCYP24 activity (e.g. transcription, translation, activity of translatedprotein, etc.) the activity level is preferably determined with respectto the VDR activity level. When a sample tissue (e.g. tissue biopsy)shows a higher level of CYP24 activity than a control sample (e.g.healthy tissue) (preferably at the same level of VDR activity) then theelevated CYP24 activity indicates the presence of, prognosis of, orpredisposition to develop, a cancer.

The VDR transcript (e.g., mRNA) levels or translated protein levels canbe measured using the assays described above for CYP24 activity; theonly difference being that the assay is adjusted for specificity to VDRnucleic acids or polypeptides rather than to CYP24.

Antibodies specific for VDR are commercially available (AffinityBioReagents #PA1-711, MA1-710, Santa Cruz Biotechnology #sc-1008,sc-1009). Gene specific probes for CYP24 and VDR mRNAs that can be usedto generate riboprobes for mRNA FISH are provided in Example 1. Inaddition, an assay for CYP24 and VDR transcription levels is illustratedin Example 1.

D) Hybridization Formats and Optimization of Hybridization Conditions.

1) Array-Based Hybridization Formats.

The methods of this invention are particularly well suited toarray-based hybridization formats. For a description of one preferredarray-based hybridization system see Pinkel et al. (1998) NatureGenetics, 20: 207-211.

Arrays are a multiplicity of different “probe” or “target” nucleic acids(or other compounds) attached to one or more surfaces (e.g., solid,membrane, or gel). In a preferred embodiment, the multiplicity ofnucleic acids (or other moieties) is attached to a single contiguoussurface or to a multiplicity of surfaces juxtaposed to each other.

In an array format a large number of different hybridization reactionscan be run essentially “in parallel.” This provides rapid, essentiallysimultaneous, evaluation of a number of hybridizations in a single“experiment”. Methods of performing hybridization reactions in arraybased formats are well known to those of skill in the art (see, e.g.,Pastinen (1997) Genome Res. 7: 606-614; Jackson (1996) NatureBiotechnology 14:1685; Chee (1995) Science 274: 610; WO 96/17958, Pinkelet al. (1998) Nature Genetics 20: 207-211).

Arrays, particularly nucleic acid arrays can be produced according to awide variety of methods well known to those of skill in the art. Forexample, in a simple embodiment, “low density” arrays can simply beproduced by spotting (e.g. by hand using a pipette) different nucleicacids at different locations on a solid support (e.g. a glass surface, amembrane, etc.).

This simple spotting, approach has been automated to produce highdensity spotted arrays (see, e.g., U.S. Pat. No. 5,807,522). This patentdescribes the use of an automated system that taps a microcapillaryagainst a surface to deposit a small volume of a biological sample. Theprocess is repeated to generate high density arrays.

Arrays can also be produced using oligonucleotide synthesis technology.Thus, for example, U.S. Pat. No. 5,143,854 and PCT Patent PublicationNos. WO 90/15070 and 92/10092 teach the use of light-directedcombinatorial synthesis of high density oligonucleotide arrays.

In brief, the light-directed combinatorial synthesis of oligonucleotidearrays on glass surfaces proceeds using automated phosphoramiditechemistry and chip masking techniques. In one specific implementation, aglass surface is derivatized with a silane reagent containing afunctional group, e.g., a hydroxyl or amine group blocked by aphotolabile protecting group. Photolysis through a photolithographicmask is used selectively to expose functional groups which are thenready to react with incoming 5′-photoprotected nucleosidephosphoramidites. The phosphoramidites react only with those sites whichare illuminated (and thus exposed by removal of the photolabile blockinggroup). Thus, the phosphoramidites only add to those areas selectivelyexposed from the preceding step. These steps are repeated until thedesired array of sequences have been synthesized on the solid surface.Combinatorial synthesis of different oligonucleotide analogues atdifferent locations on the array is determined by the pattern ofillumination during synthesis and the order of addition of couplingreagents.

In a preferred embodiment, the arrays used in this invention cancomprise either probe or target nucleic acids. These probes or targetnucleic acids are then hybridized respectively with their “target”nucleic acids. Because the CYP24 gene sequence is known, oligonucleotidearrays can be synthesized containing one or multiple probes specific toCYP24.

In another embodiment the array, particularly a spotted array, caninclude genomic DNA, e.g. overlapping clones that provide a highresolution scan of the amplicon containing to CYP24, or of CYP24 itself.Amplicon nucleic acid can be obtained from, e.g., HACs, MACs, YACs,BACs, PACs, P1s, cosmids, plasmids, inter-Alu PCR products of genomicclones, restriction digests of genomic clones, cDNA clones,amplification (e.g., PCR) products, and the like.

In various embodiments, the array nucleic acids are derived frompreviously mapped libraries of clones spanning or including the ampliconsequences of the invention, as well as clones from other areas of thegenome, as described below. The arrays can be hybridized with a singlepopulation of sample nucleic acid or can be used with two differentiallylabeled collections (as with an test sample and a reference sample).

Many methods for immobilizing nucleic acids on a variety of solidsurfaces are known in the art. A wide variety of organic and inorganicpolymers, as well as other materials, both natural and synthetic, can beemployed as the material for the solid surface. Illustrative solidsurfaces include, e.g., nitrocellulose, nylon, glass, quartz, diazotizedmembranes (paper or nylon), silicones, polyformaldehyde, cellulose, andcellulose acetate. In addition, plastics such as polyethylene,polypropylene, polystyrene, and the like can be used. Other materialswhich may be employed include paper, ceramics, metals, metalloids,semiconductive materials, cermets or the like. In addition, substancesthat form gels can be used. Such materials include, e.g., proteins(e.g., gelatins), lipopolysaccharides, silicates, agarose andpolyacrylamides. Where the solid surface is porous, various pore sizesmay be employed depending upon the nature of the system.

In preparing the surface, a plurality of different materials may beemployed, particularly as laminates, to obtain various properties. Forexample, proteins (e.g., bovine serum albumin) or mixtures ofmacromolecules (e.g., Denhardt's solution) can be employed to avoidnon-specific binding, simplify covalent conjugation, enhance signaldetection or the like. If covalent bonding between a compound and thesurface is desired, the surface will usually be polyfunctional or becapable of being polyfunctionalized. Functional groups which may bepresent on the surface and used for linking can include carboxylicacids, aldehydes, amino groups, cyano groups, ethylenic groups, hydroxylgroups, mercapto groups and the like. The manner of linking a widevariety of compounds to various surfaces is well known and is amplyillustrated in the literature.

For example, methods for immobilizing nucleic acids by introduction ofvarious functional groups to the molecules is known (see, e.g., Bischoff(1987) Anal. Biochem., 164: 336-344; Kremsky (1987) Nucl. Acids Res. 15:2891-2910). Modified nucleotides can be placed on the target using PCRprimers containing the modified nucleotide, or by enzymatic end labelingwith modified nucleotides. Use of glass or membrane supports (e.g.,nitrocellulose, nylon, polypropylene) for the nucleic acid arrays of theinvention is advantageous because of well developed technology employingmanual and robotic methods of arraying targets at relatively highelement densities. Such membranes are generally available and protocolsand equipment for hybridization to membranes is well known.

Target elements of various sizes, ranging from 1 mm diameter down to 1μm can be used. Smaller target elements containing low amounts ofconcentrated, fixed probe DNA are used for high complexity comparativehybridizations since the total amount of sample available for binding toeach target element will be limited. Thus it is advantageous to havesmall array target elements that contain a small amount of concentratedprobe DNA so that the signal that is obtained is highly localized andbright. Such small array target elements are typically used in arrayswith densities greater than 10⁴/cm². Relatively simple approachescapable of quantitative fluorescent imaging of 1 cm² areas have beendescribed that permit acquisition of data from a large number of targetelements in a single image (see, e.g., Wittrup (1994) Cytometry16:206-213, Pinkel et al. (1998) Nature Genetics 20: 207-211).

Arrays on solid surface substrates with much lower fluorescence thanmembranes, such as glass, quartz, or small beads, can achieve muchbetter sensitivity. Substrates such as glass or fused silica areadvantageous in that they provide a very low fluorescence substrate, anda highly efficient hybridization environment. Covalent attachment of thetarget nucleic acids to glass or synthetic fused silica can beaccomplished according to a number of known techniques (describedabove). Nucleic acids can be conveniently coupled to glass usingcommercially available reagents. For instance, materials for preparationof silanized glass with a number of functional groups are commerciallyavailable or can be prepared using standard techniques (see, e.g., Gait(1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press,Wash., D.C.). Quartz cover slips, which have at least 10-fold lowerautofluorescence than glass, can also be silanized.

Alternatively, probes can also be immobilized on commercially availablecoated beads or other surfaces. For instance, biotin end-labeled nucleicacids can be bound to commercially available avidin-coated beads.Streptavidin or anti-digoxigenin antibody can also be attached tosilanized glass slides by protein-mediated coupling using e.g., proteinA following standard protocols (see, e.g., Smith (1992) Science 258:1122-1126). Biotin or digoxigenin end-labeled nucleic acids can beprepared according to standard techniques. Hybridization to nucleicacids attached to beads is accomplished by suspending them in thehybridization mix, and then depositing them on the glass substrate foranalysis after washing. Alternatively, paramagnetic particles, such asferric oxide particles, with or without avidin coating, can be used.

In one particularly preferred embodiment, probe nucleic acid is spottedonto a surface (e.g., a glass or quartz surface). The nucleic acid isdissolved in a mixture of water, dimethylsulfoxide (DMSO), andnitrocellulose and spotted onto amino-silane coated glass slides. Smallcapillaries tubes can be used to “spot” the probe mixture.

2) Other Hybridization Formats.

A variety of nucleic acid hybridization formats are known to thoseskilled in the art. For example, common formats include sandwich assaysand competition or displacement assays. Hybridization techniques aregenerally described in Hames and Higgins (1985) Nucleic AcidHybridization, A Practical Approach, IRL Press; Gall and Pardue (1969)Proc. Natl. Acad. Sci. USA 63: 378-383; and John et al. (1969) Nature223: 582-587.

Sandwich assays are commercially useful hybridization assays fordetecting or isolating nucleic acid sequences. Such assays utilize a“capture” nucleic acid covalently immobilized to a solid support and alabeled “signal” nucleic acid in solution. The sample will provide thetarget nucleic acid. The “capture” nucleic acid and “signal” nucleicacid probe hybridize with the target nucleic acid to form a “sandwich”hybridization complex. To be most effective, the signal nucleic acidshould not hybridize with the capture nucleic acid.

Typically, labeled signal nucleic acids are used to detecthybridization. Complementary nucleic acids or signal nucleic acids maybe labeled by any one of several methods typically used to detect thepresence of hybridized polynucleotides. The most common method ofdetection is the use of autoradiography with ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P-labelled probes or the like. Other labels include ligands that bindto labeled antibodies, fluorophores, chemi-luminescent agents, enzymes,and antibodies which can serve as specific binding pair members for alabeled ligand.

Detection of a hybridization complex may require the binding of a signalgenerating complex to a duplex of target and probe polynucleotides ornucleic acids. Typically, such binding occurs through ligand andanti-ligand interactions as between a ligand-conjugated probe and ananti-ligand conjugated with a signal.

The sensitivity of the hybridization assays may be enhanced through useof a nucleic acid amplification system that multiplies the targetnucleic acid being detected. Examples of such systems include thepolymerase chain reaction (PCR) system and the ligase chain reaction(LCR) system. Other methods recently described in the art are thenucleic acid sequence based amplification (NASBAO, Cangene, Mississauga,Ontario) and Q Beta Replicase systems.

3) Optimization of Hybridization Conditions.

Nucleic acid hybridization simply involves providing a denatured probeand target nucleic acid under conditions where the probe and itscomplementary target can form stable hybrid duplexes throughcomplementary base pairing. The nucleic acids that do not form hybridduplexes are then washed away leaving the hybridized nucleic acids to bedetected, typically through detection of an attached detectable label.It is generally recognized that nucleic acids are denatured byincreasing the temperature or decreasing the salt concentration of thebuffer containing the nucleic acids, or in the addition of chemicalagents, or the raising of the pH. Under low stringency conditions (e.g.,low temperature and/or high salt and/or high target concentration)hybrid duplexes (e.g., DNA:DNA, RNA:RNA, or RNA:DNA) will form evenwhere the annealed sequences are not perfectly complementary. Thusspecificity of hybridization is reduced at lower stringency. Conversely,at higher stringency (e.g., higher temperature or lower salt) successfulhybridization requires fewer mismatches.

One of skill in the art will appreciate that hybridization conditionsmay be selected to provide any degree of stringency. In a preferredembodiment, hybridization is performed at low stringency to ensurehybridization and then subsequent washes are performed at higherstringency to eliminate mismatched hybrid duplexes. Successive washesmay be performed at increasingly higher stringency (e.g., down to as lowas 0.25×SSPE at 37° C. to 70° C.) until a desired level of hybridizationspecificity is obtained. Stringency can also be increased by addition ofagents such as formamide. Hybridization specificity may be evaluated bycomparison of hybridization to the test probes with hybridization to thevarious controls that can be present.

In general, there is a tradeoff between hybridization specificity(stringency) and signal intensity. Thus, in a preferred embodiment, thewash is performed at the highest stringency that produces consistentresults and that provides a signal intensity greater than approximately10% of the background intensity. Thus, in a preferred embodiment, thehybridized array may be washed at successively higher stringencysolutions and read between each wash. Analysis of the data sets thusproduced will reveal a wash stringency above which the hybridizationpattern is not appreciably altered and which provides adequate signalfor the particular probes of interest.

In a preferred embodiment, background signal is reduced by the use of adetergent (e.g., C-TAB) or a blocking reagent (e.g., tRNA, sperm DNA,cot-1 DNA, etc.) during the hybridization to reduce non-specificbinding. In a particularly preferred embodiment, the hybridization isperformed in the presence of about 10 μg/1 μL tRNA. The use of blockingagents in hybridization is well known to those of skill in the art (see,e.g., Chapter 8 in P. Tijssen, supra.)

Methods of optimizing hybridization conditions are well known to thoseof skill in the art (see, e.g., Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology, Vol. 24: Hybridization With NucleicAcid Probes, Elsevier, N.Y.).

Optimal conditions are also a function of the sensitivity of label(e.g., fluorescence) detection for different combinations of substratetype, fluorochrome, excitation and emission bands, spot size and thelike. Low fluorescence background surfaces can be used (see, e.g., Chu(1992) Electrophoresis 13:105-114). The sensitivity for detection ofspots (“target elements”) of various diameters on the candidate surfacescan be readily determined by, e.g., spotting a dilution series offluorescently end labeled DNA fragments. These spots are then imagedusing conventional fluorescence microscopy. The sensitivity, linearity,and dynamic range achievable from the various combinations offluorochrome and solid surfaces (e.g., glass, fused silica, etc.) canthus be determined. Serial dilutions of pairs of fluorochrome in knownrelative proportions can also be analyzed. This determines the accuracywith which fluorescence ratio measurements reflect actual fluorochromeratios over the dynamic range permitted by the detectors andfluorescence of the substrate upon which the probe has been fixed.

4) Labeling and Detection of Nucleic Acids.

In a preferred embodiment, the hybridized nucleic acids are detected bydetecting one or more labels attached to the sample nucleic acids. Thelabels may be incorporated by any of a number of means well known tothose of skill in the art. Means of attaching labels to nucleic acidsinclude, for example nick translation, or end-labeling by kinasing ofthe nucleic acid and subsequent attachment (ligation) of a linkerjoining the sample nucleic acid to a label (e.g., a fluorophore). A widevariety of linkers for the attachment of labels to nucleic acids arealso known. In addition, intercalating dyes and fluorescent nucleotidescan also be used.

Detectable labels suitable for use in the present invention include anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include biotin for staining with labeledstreptavidin conjugate, magnetic beads (e.g., Dynabeads™), fluorescentdyes (e.g., fluorescein, texas red, rhodamine, green fluorescentprotein, and the like, see, e.g., Molecular Probes, Eugene, Oreg., USA),radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase and others commonly used in anELISA), and colorimetric labels such as colloidal gold (e.g., goldparticles in the 40-80 nm diameter size range scatter green light withhigh efficiency) or colored glass or plastic (e.g., polystyrene,polypropylene, latex, etc.) beads. Patents teaching the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149; and 4,366,241.

A fluorescent label is preferred because it provides a very strongsignal with low background. It is also optically detectable at highresolution and sensitivity through a quick scanning procedure. Thenucleic acid samples can all be labeled with a single label, e.g., asingle fluorescent label. Alternatively, in another embodiment,different nucleic acid samples can be simultaneously hybridized whereeach nucleic acid sample has a different label. For instance, one targetcould have a green fluorescent label and a second target could have ared fluorescent label. The scanning step will distinguish sites ofbinding of the red label from those binding the green fluorescent label.Each nucleic acid sample (target nucleic acid) can be analyzedindependently from one another.

Suitable chromogens which can be employed include those molecules andcompounds which absorb light in a distinctive range of wavelengths sothat a color can be observed or, alternatively, which emit light whenirradiated with radiation of a particular wave length or wave lengthrange, e.g., fluorescers.

Desirably, fluorescers should absorb light above about 300 nm,preferably about 350 nm, and more preferably above about 400 nm, usuallyemitting at wavelengths greater than about 10 nm higher than thewavelength of the light absorbed. It should be noted that the absorptionand emission characteristics of the bound dye can differ from theunbound dye. Therefore, when referring to the various wavelength rangesand characteristics of the dyes, it is intended to indicate the dyes asemployed and not the dye which is unconjugated and characterized in anarbitrary solvent.

Fluorescers are generally preferred because by irradiating a fluorescerwith light, one can obtain a plurality of emissions. Thus, a singlelabel can provide for a plurality of measurable events.

Detectable signal can also be provided by chemiluminescent andbioluminescent sources. Chemiluminescent sources include a compoundwhich becomes electronically excited by a chemical reaction and can thenemit light which serves as the detectable signal or donates energy to afluorescent acceptor. Alternatively, luciferins can be used inconjunction with luciferase or lucigenins to provide bioluminescence.

Spin labels are provided by reporter molecules with an unpaired electronspin which can be detected by electron spin resonance (ESR)spectroscopy. Exemplary spin labels include organic free radicals,transitional metal complexes, particularly vanadium, copper, iron, andmanganese, and the like. Exemplary spin labels include nitroxide freeradicals.

The label may be added to the target (sample) nucleic acid(s) prior to,or after the hybridization. So called “direct labels” are detectablelabels that are directly attached to or incorporated into the target(sample) nucleic acid prior to hybridization. In contrast, so called“indirect labels” are joined to the hybrid duplex after hybridization.Often, the indirect label is attached to a binding moiety that has beenattached to the target nucleic acid prior to the hybridization. Thus,for example, the target nucleic acid may be biotinylated before thehybridization. After hybridization, an avidin-conjugated fluorophorewill bind the biotin bearing hybrid duplexes providing a label that iseasily detected. For a detailed review of methods of labeling nucleicacids and detecting labeled hybridized nucleic acids see LaboratoryTechniques in Biochemistry and Molecular Biology, Vol. 24: HybridizationWith Nucleic Acid Probes, P. Tijssen, ed. Elsevier, N.Y., (1993)).

Fluorescent labels are easily added during an in vitro transcriptionreaction. Thus, for example, fluorescein labeled UTP and CTP can beincorporated into the RNA produced in an in vitro transcription.

The labels can be attached directly or through a linker moiety. Ingeneral, the site of label or linker-label attachment is not limited toany specific position. For example, a label may be attached to anucleoside, nucleotide, or analogue thereof at any position that doesnot interfere with detection or hybridization as desired. For example,certain Label-ON Reagents from Clontech (Palo Alto, Calif.) provide forlabeling interspersed throughout the phosphate backbone of anoligonucleotide and for terminal labeling at the 3′ and 5′ ends. Asshown for example herein, labels can be attached at positions on theribose ring or the ribose can be modified and even eliminated asdesired. The base moieties of useful labeling reagents can include thosethat are naturally occurring or modified in a manner that does notinterfere with the purpose to which they are put. Modified bases includebut are not limited to 7-deaza A and G, 7-deaza-8-aza A and G, and otherheterocyclic moieties.

It will be recognized that fluorescent labels are not to be limited tosingle species organic molecules, but include inorganic molecules,multi-molecular mixtures of organic and/or inorganic molecules,crystals, heteropolymers, and the like. Thus, for example, CdSe—CdScore-shell nanocrystals enclosed in a silica shell can be easilyderivatized for coupling to a biological molecule (Bruchez et al. (1998)Science, 281: 2013-2016). Similarly, highly fluorescent quantum dots(zinc sulfide-capped cadmium selenide) have been covalently coupled tobiomolecules for use in ultrasensitive biological detection (Warren andNie (1998) Science, 281: 2016-2018).

E) Antibodies to CYP24.

Either polyclonal or monoclonal antibodies may be used in theimmunoassays and therapeutic methods of the invention described herein.Polyclonal antibodies are preferably raised by multiple injections (e.g.subcutaneous or intramuscular injections) of substantially pure CYP24polypeptides or antigenic CYP24 polypeptides into a suitable non-humanmammal. The antigenicity of CYP24 peptides can be determined byconventional techniques to determine the magnitude of the antibodyresponse of an animal that has been immunized with the peptide.Generally, the CYP24 peptides that are used to raise the anti-CYP24antibodies should generally be those which induce production of hightiters of antibody with relatively high affinity for CYP24.

If desired, the immunizing peptide may be coupled to a carrier proteinby conjugation using techniques which are well-known in the art. Suchcommonly used carriers which are chemically coupled to the peptideinclude keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serumalbumin (BSA), and tetanus toxoid. The coupled peptide is then used toimmunize the animal (e.g. a mouse or a rabbit). Because CYP24 may beconserved among mammalian species, use of a carrier protein to enhancethe immunogenicity of CYP24 proteins is preferred.

The antibodies are then obtained from blood samples taken from themammal. The techniques used to develop polyclonal antibodies are knownin the art (see, e.g., Methods of Enzymology, “Production of AntiseraWith Small Doses of Immunogen: Multiple Intradermal Injections”,Langone, et al. eds. (Acad. Press, 1981)). Polyclonal antibodiesproduced by the animals can be further purified, for example, by bindingto and elution from a matrix to which the peptide to which theantibodies were raised is bound. Those of skill in the art will know ofvarious techniques common in the immunology arts for purification and/orconcentration of polyclonal antibodies, as well as monoclonal antibodiessee, for example, Coligan, et al. (1991) Unit 9, Current Protocols inImmunology, Wiley Interscience).

Preferably, however, the CYP24 antibodies produced will be monoclonalantibodies (“mAb's”). For preparation of monoclonal antibodies,immunization of a mouse or rat is preferred. The term “antibody” as usedin this invention includes intact molecules as well as fragmentsthereof, such as, Fab and F(ab′)^(2′) which are capable of binding anepitopic determinant. Also, in this context, the term “mab's of theinvention” refers to monoclonal antibodies with specificity for CYP24.

The general method used for production of hybridomas secreting mAbs iswell known (Kohler and Milstein (1975) Nature, 256:495). Briefly, asdescribed by Kohler and Milstein the technique comprised isolatinglymphocytes from regional draining lymph nodes of five separate cancerpatients with either melanoma, teratocarcinoma or cancer of the cervix,glioma or lung, (where samples were obtained from surgical specimens),pooling the cells, and fusing the cells with SHFP-1. Hybridomas werescreened for production of antibody which bound to cancer cell lines.

Confirmation of CYP24 specificity among mAb's can be accomplished usingrelatively routine screening techniques (such as the enzyme-linkedimmunosorbent assay, or “ELISA”) to determine the elementary reactionpattern of the mAb of interest.

It is also possible to evaluate an mAb to determine whether it has thesame specificity as a mAb of the invention without undue experimentationby determining whether the mAb being tested prevents a mAb of theinvention from binding to CYP24 isolated as described above. If the mAbbeing tested competes with the mAb of the invention, as shown by adecrease in binding by the mAb of the invention, then it is likely thatthe two monoclonal antibodies bind to the same or a closely relatedepitope. Still another way to determine whether a mAb has thespecificity of a mAb of the invention is to preincubate the mAb of theinvention with an antigen with which it is normally reactive, anddetermine if the mAb being tested is inhibited in its ability to bindthe antigen. If the mAb being tested is inhibited then, in alllikelihood, it has the same, or a closely related, epitopic specificityas the mAb of the invention.

Antibodies fragments, e.g. single chain antibodies (scFv or others), canalso be produced/selected using phage display technology. The ability toexpress antibody fragments on the surface of viruses that infectbacteria (bacteriophage or phage) makes it possible to isolate a singlebinding antibody fragment from a library of greater than 10¹⁰ nonbindingclones. To express antibody fragments on the surface of phage (phagedisplay), an antibody fragment gene is inserted into the gene encoding aphage surface protein (pIII) and the antibody fragment-pIII fusionprotein is displayed on the phage surface (McCafferty et al. (1990)Nature, 348: 552-554; Hoogenboom et al. (1991) Nucleic Acids Res. 19:4133-4137).

Since the antibody fragments on the surface of the phage are functional,phage bearing antigen binding antibody fragments can be separated fromnon-binding phage by antigen affinity chromatography (McCafferty et al.(1990) Nature, 348: 552-554). Depending on the affinity of the antibodyfragment, enrichment factors of 20 fold-1,000,000 fold are obtained fora single round of affinity selection. By infecting bacteria with theeluted phage, however, more phage can be grown and subjected to anotherround of selection. In this way, an enrichment of 1000 fold in one roundcan become 1,000,000 fold in two rounds of selection (McCafferty et al.(1990) Nature, 348: 552-554). Thus even when enrichments are low (Markset al. (1991) J. Mol. Biol. 222: 581-597), multiple rounds of affinityselection can lead to the isolation of rare phage. Since selection ofthe phage antibody library on antigen results in enrichment, themajority of clones bind antigen after as few as three to four rounds ofselection. Thus only a relatively small number of clones (severalhundred) need to be analyzed for binding to antigen.

Human antibodies can be produced without prior immunization bydisplaying very large and diverse V-gene repertoires on phage (Marks etal. (1991) J. Mol. Biol. 222: 581-597). In one embodiment natural V_(H)and V_(L) repertoires present in human peripheral blood lymphocytes arewere isolated from unimmunized donors by PCR. The V-gene repertoireswere spliced together at random using PCR to create a scFv generepertoire which is was cloned into a phage vector to create a libraryof 30 million phage antibodies (Id.). From this single “naive” phageantibody library, binding antibody fragments have been isolated againstmore than 17 different antigens, including haptens, polysaccharides andproteins (Marks et al. (1991) J. Mol. Biol. 222: 581-597; Marks et al.(1993). Bio/Technology. 10: 779-783; Griffiths et al. (1993) EMBO J. 12:725-734; Clackson et al. (1991) Nature. 352: 624-628). Antibodies havebeen produced against self proteins, including human thyroglobulin,immunoglobulin, tumor necrosis factor and CEA (Griffiths et al. (1993)EMBO J. 12: 725-734). It is also possible to isolate antibodies againstcell surface antigens by selecting directly on intact cells. Theantibody fragments are highly specific for the antigen used forselection and have affinities in the 1:M to 100 nM range (Marks et al.(1991) J. Mol. Biol. 222: 581-597; Griffiths et al. (1993) EMBO J. 12:725-734). Larger phage antibody libraries result in the isolation ofmore antibodies of higher binding affinity to a greater proportion ofantigens.

It will also be recognized that CYP24 antibodies can be prepared by anyof a number of commercial services (e.g., Berkeley antibodylaboratories, Bethyl Laboratories, Anawa, Eurogenetec, etc.).

II. Assay Optimization—Determining Prognostically Significant Levels.

The assays of this invention have immediate utility indetecting/predicting the likelihood of a cancer, in estimating survivalfrom a cancer, in screening for agents that modulate CYP24 activity, andin screening for agents that inhibit cell proliferation. In particular,for example, identification of an amplification in CYP24 (genomic DNA)indicates the presence of a cancer and/or the predisposition to developa cancer.

Methods of optimizing predictive/diagnostic assays are well known tothose of ordinary skill in the art. Typically this involves determining“baseline levels” (e.g. of CYP24) in normal tissues and CYP24 activitylevels in pathological (i.e. tumor tissues). In particularly preferredembodiments, such levels are determined with appropriate controls forconcurrent VDR activity, sample type, age, sex, developmental state,overall physiological state (e.g. in a non-pregnant as compared to apregnant female), overall health, tumor type, etc. In a preferredembodiment, “baseline” (e.g., control) levels are determined from anormal (healthy) tissue from the same individual or from individuals ofthe same population. Alternatively, “baseline” and “pathological” levelsare determined from “population” studies” that provide sufficient samplesize and diversity that the influence of the various co-factorsidentified above (age, health, sex, etc.) can be of statisticallyevaluated. “Baseline” CYP24 levels can also be evaluated by reference tomodel systems, e.g., as described in Examples 3-5.

In a preferred embodiment, quantitative assays of CYP24 level are deemedto show a positive result, e.g. elevated CYP24 level, when the measuredCYP24 level is greater than the level measured or known for a controlsample (e.g. either a level known or measured for a normal healthymammal of the same species or a “baseline/reference” level determined ata different tissue and/or a different time for the same individual. In aparticularly preferred embodiment, the assay is deemed to show apositive result (e.g., “a prognostically significant level”) when thedifference between sample and “control” is statistically significant(e.g. at the 85% or greater, preferably at the 90% or greater, morepreferably at the 95% or greater and most preferably at the 98% orgreater confidence level).

III. Methods of Treating Cancer—Selection of Adjuvant Therapy Based onCYP24 Level.

Because of the ability to evaluate the presence of, or thepredisposition to develop, a cancer, the assays of this invention make auseful component of a cancer therapy regimen. Thus, in one embodiment,CYP24 activity can be used as a measure of disease progression, while inanother embodiment CYP24 activity is used to evaluate the necessity ofan adjuvant therapy.

“Adjuvant cancer therapy” refers to a method of treating cancer, such aschemotherapy, radiation therapy, surgery, reoperation, antihormonetherapy, and immunotherapy, that is administered in combination with orfollowing another method of cancer treatment. An “adjuvant cancertherapy” often represents an aggressive form of cancer treatment that isselected in view of a reduced survival expectancy and/or a detectedlevel of CYP24 that is elevated compared to a control level.

Adjuvant therapies are well known to those of skill in the art andinclude, but are not limited to chemotherapy, radiation therapy, primarysurgery or reoperation, antihormone therapy, immunotherapy, and thelike. “Chemotherapy”, as used in this context, refers to theadministration of chemical compounds to an animal with cancer that isaimed at killing or reducing the number of cancer cells within theanimal. Generally, chemotherapeutic agents arrest the growth of or killcells that are dividing or growing, such as cancer cells.Chemotherapeutic agents for use against cancer are well known to thoseof skill in the art include, but are not limited to doxirubicin,vinblastine, genistein, etc.

“Radiation therapy” in this context refers to the administration ofradioactivity to an animal with cancer. Radiation kills or inhibits thegrowth of dividing cells, such as cancer cells. The administration maybe by an external source (e.g., a gamma source, a proton source, amolecular beam source, etc.) or may be by an implantable radioactivematerial. Radiation therapy includes “traditional” radiation treatmentaimed at reduction or elimination of tumor volume or more aggressiveradio-surgery techniques.

Surgical methods refer to the direct removal or ablation of cells, e.g.cancer cells, from an animal. Most often, the cancer cells will be inthe form of a tumor (e.g. a mammary tumor), which is removed from theanimal. The surgical methods may involve removal of healthy as well aspathological tissue. “Reoperation” refers to surgery performed on ananimal that has previously undergone surgery for treatment of the samepathology.

“Antihormone therapy” refers to the administration of compounds thatcounteract or inhibit hormones, such as estrogen or androgen, that havea mitogenic effect on cells. Often, these hormones act to increase thecancerous properties of cancer cells in vivo.

Immunotherapy refers to methods of enhancing the ability of an animal'simmune system to destroy cancer cells within the animal. This caninvolve the treatment with polyclonal or monoclonal antibodies that bindparticular tumor-specific markers (e.g. IL-13 receptor, and Lewis Y(Le^(Y)) marker, etc.) help to direct cytotoxins of native immune systemeffectors to the tumor target. Immunotherapeutic methods are well knowto those of skill in the art (see, e.g., Pastan et al. (1992) Ann. Rev.Biochem., 61: 331-354, Brinkman and Pastan (1994) Biochimica BiphysicaActa, 1198: 27-45, etc.).

IV. Screening for Therapeutics

It was also a discovery of this invention that downregulation of CYP24activity (at a given level of vitamin D receptor activity) is expectedto act prophylactically to prevent the development of cancers and/or toact therapeutically to reduce or eliminate a cancer. Thus, in oneembodiment, this invention provides methods of screening for agents thatmodulate and preferably that down regulate CYP24 activity.Downregulation, as used in this context, includes decrease in CYP24transcription and/or decrease in CYP24 translation, and/or decrease inCYP24 polypeptide activity.

Preferred “screening” methods of this invention involve contacting aCYP24-expressing cell (e.g., a cell capable of expressing CYP24) with atest agent; and (ii) detecting the level of CYP24 activity (e.g. asdescribed above), where a decreased level of CYP24 activity as comparedto the level of CYP24 activity in a cell not contacted with the agentindicates that said agent inhibits or downregulates CYP24 and/orinhibits proliferation of the cell.

Virtually any agent can be tested in such an assay. Such agents include,but are not limited to natural or synthetic nucleic acids, natural orsynthetic polypeptides, natural or synthetic lipids, natural orsynthetic small organic molecules, and the like. In one preferredformat, test agents are provided as members of a combinatorial library.

A) Combinatorial Libraries (e.g., Small Organic Molecules).

Conventionally, new chemical entities with useful properties aregenerated by identifying a chemical compound (called a “lead compound”)with some desirable property or activity, creating variants of the leadcompound, and evaluating the property and activity of those variantcompounds. However, the current trend is to shorten the time scale forall aspects of drug discovery. Because of the ability to test largenumbers quickly and efficiently, high throughput screening (HTS) methodsare replacing conventional lead compound identification methods.

In one preferred embodiment, high throughput screening methods involveproviding a library containing a large number of potential therapeuticcompounds (candidate compounds). Such “combinatorial chemical libraries”are then screened in one or more assays, as described below to identifythose library members (particular chemical species or subclasses) thatdisplay a desired characteristic activity. The compounds thus identifiedcan serve as conventional “lead compounds” or can themselves be used aspotential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biological synthesisby combining a number of chemical “building blocks” such as reagents.For example, a linear combinatorial chemical library such as apolypeptide (e.g., mutein) library is formed by combining a set ofchemical building blocks called amino acids in every possible way for agiven compound length (i.e., the number of amino acids in a polypeptidecompound). Millions of chemical compounds can be synthesized throughsuch combinatorial mixing of chemical building blocks. For example, onecommentator has observed that the systematic, combinatorial mixing of100 interchangeable chemical building blocks results in the theoreticalsynthesis of 100 million tetrameric compounds or 10 billion pentamericcompounds (Gallop et al. (1994) 37(9): 1233-1250).

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka (1991) Int. J. Pept. Prot. Res., 37:487-493, Houghton et al. (1991) Nature, 354: 84-88). Peptide synthesisis by no means the only approach envisioned and intended for use withthe present invention. Other chemistries for generating chemicaldiversity libraries can also be used. Such chemistries include, but arenot limited to: peptoids (PCT Publication No WO 91/19735, 26 Dec. 1991),encoded peptides (PCT Publication WO 93/20242, 14 Oct. 1993), randombio-oligomers (PCT Publication WO 92/00091, 9 Jan. 1992),benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such ashydantoins, benzodiazepines and dipeptides (Hobbs et al., (1993) Proc.Nat. Acad. Sci. USA 90: 6909-6913), vinylogous polypeptides (Hagihara etal. (1992) J. Amer. Chem. Soc. 114: 6568), nonpeptidal peptidomimeticswith a Beta-D-Glucose scaffolding (Hirschmann et al., (1992) J. Amer.Chem. Soc. 114: 9217-9218), analogous organic syntheses of smallcompound libraries (Chen et al. (1994) J. Amer. Chem. Soc. 116: 2661),oligocarbamates (Cho, et al., (1993) Science 261:1303), and/or peptidylphosphonates (Campbell et al., (1994) J. Org. Chem. 59: 658). See,generally, Gordon et al., (1994) J. Med. Chem. 37:1385, nucleic acidlibraries (see, e.g., Strategene, Corp.), peptide nucleic acid libraries(see, e.g., U.S. Pat. No. 5,539,083) antibody libraries (see, e.g.,Vaughn et al. (1996) Nature Biotechnology, 14(3): 309-314), andPCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al. (1996)Science, 274: 1520-1522, and U.S. Pat. No. 5,593,853), and small organicmolecule libraries (see, e.g., benzodiazepines, Baum (1993) C&EN,January 18, page 33, isoprenoids U.S. Pat. No. 5,569,588,thiazolidinones and metathiazanones U.S. Pat. No. 5,549,974,pyrrolidines U.S. Pat. Nos. 5,525,735 and 5,519,134, morpholinocompounds U.S. Pat. No. 5,506,337, benzodiazepines U.S. Pat. No.5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.).

A number of well known robotic systems have also been developed forsolution phase chemistries. These systems include automated workstationslike the automated synthesis apparatus developed by Takeda ChemicalIndustries, LTD. (Osaka, Japan) and many robotic systems utilizingrobotic arms (Zymate II, Zymark Corporation, Hopkinton, Mass.; Orca,Hewlett-Packard, Palo Alto, Calif.) which mimic the manual syntheticoperations performed by a chemist. Any of the above devices are suitablefor use with the present invention. The nature and implementation ofmodifications to these devices (if any) so that they can operate asdiscussed herein will be apparent to persons skilled in the relevantart. In addition, numerous combinatorial libraries are themselvescommercially available (see, e.g., ComGenex, Princeton, N.J., Asinex,Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3DPharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).

B) High Throughput Screening

Any of the assays for compounds modulating CYP24 level described hereinare amenable to high throughput screening. Preferred assays thus detectenhancement or inhibition of CYP24 gene transcription, inhibition orenhancement of CYP24 polypeptide expression, and inhibition orenhancement of CYP24 polypeptide activity, (at a given VDR activitylevel).

High throughput assays for the presence, absence, or quantification ofparticular nucleic acids or protein products are well known to those ofskill in the art. Similarly, binding assays and reporter gene assays aresimilarly well known. Thus, for example, U.S. Pat. No. 5,559,410discloses high throughput screening methods for proteins, U.S. Pat. No.5,585,639 discloses high throughput screening methods for nucleic acidbinding (i.e., in arrays), while U.S. Pat. Nos. 5,576,220 and 5,541,061disclose high throughput methods of screening for ligand/antibodybinding.

In addition, high throughput screening systems are commerciallyavailable (see, e.g., Zymark Corp., Hopkinton, Mass.; Air TechnicalIndustries, Mentor, Ohio; Beckman Instruments, Inc. Fullerton, Calif.;Precision Systems, Inc., Natick, Mass., etc.). These systems typicallyautomate entire procedures including all sample and reagent pipetting,liquid dispensing, timed incubations, and final readings of themicroplate in detector(s) appropriate for the assay. These configurablesystems provide high throughput and rapid start up as well as a highdegree of flexibility and customization. The manufacturers of suchsystems provide detailed protocols for various high throughput systems.Thus, for example, Zymark Corp. provides technical bulletins describingscreening systems for detecting the modulation of gene transcription,ligand binding, and the like.

V. Reducing CYP24 Activity Levels in Cells

In another embodiment, this invention provides methods of reducing CYP24activity levels in a cell. In this context, a reduction of CYP24activity is a decrease in CYP24 activity as compared to the same cell inan “untreated” condition. More preferably as compared to the same cellat the same level of VDR activity or normalized for VDR activity.

Methods of reducing activity levels of a particular gene or gene productare well known to those of skill in the art. Such methods include, butare not limited to targeting transcription or translation, e.g. by theuse of antisense molecules or ribozymes, by targeting transcriptionfactors, e.g. with antibodies or DNA binding proteins, and by targetingthe polypeptide product, e.g. by competition with inactive bindingagents (e.g. muteins), by direct blocking, e.g. by binding withantibodies or other ligands, etc.

A) Antisense Molecules.

CYP24 activity can be downregulated, or entirely inhibited, by the useof antisense molecules. An “antisense sequence or antisense nucleicacid” is a nucleic acid is complementary to the coding CYP24 mRNAnucleic acid sequence or a subsequence thereof. Binding of the antisensemolecule to the CYP24 mRNA interferes with normal translation of theCYP24 polypeptide.

Thus, in accordance with preferred embodiments of this invention,preferred antisense molecules include nucleic acids (e.g.oligonucleotides and oligonucleotide analogs) that are hybridizable withCYP24 messenger RNA. This relationship is commonly denominated as“antisense.” The antisense nucleic acids analogs are able to inhibit thefunction of the RNA, either its translation into protein, itstranslocation into the cytoplasm, or any other activity necessary to itsoverall biological function. The failure of the messenger RNA to performall or part of its function results in a reduction or completeinhibition of expression of CYP24 polypeptides.

In the context of this invention, the term “oligonucleotide” refers to apolynucleotide formed from naturally-occurring bases and/orcyclofuranosyl groups joined by native phosphodiester bonds. This termeffectively refers to naturally-occurring species or synthetic speciesformed from naturally-occurring subunits or their close homologs. Theterm “oligonucleotide” may also refer to moieties which functionsimilarly to oligonucleotides, but which have non naturally-occurringportions. Thus, oligonucleotides may have altered sugar moieties orinter-sugar linkages. Exemplary among these are the phosphorothioate andother sulfur containing species which are known for use in the art. Inaccordance with some preferred embodiments, at least one of thephosphodiester bonds of the oligonucleotide has been substituted with astructure which functions to enhance the ability of the compositions topenetrate into the region of cells where the RNA whose activity is to bemodulated is located. It is preferred that such substitutions comprisephosphorothioate bonds, methyl phosphonate bonds, or short chain alkylor cycloalkyl structures. In accordance with other preferredembodiments, the phosphodiester bonds are substituted with structureswhich are, at once, substantially non-ionic and non-chiral, or withstructures which are chiral and enantiomerically specific. Persons ofordinary skill in the art will be able to select other linkages for usein the practice of the invention.

Oligonucleotides may also include species that include at least somemodified base forms. Thus, purines and pyrimidines other than thosenormally found in nature may be so employed. Similarly, modifications onthe furanosyl portions of the nucleotide subunits may also be effected,as long as the essential tenets of this invention are adhered to.Examples of such modifications are 2′-O-alkyl- and2′-halogen-substituted nucleotides. Some specific examples ofmodifications at the 2′ position of sugar moieties which are useful inthe present invention are OH, SH, SCH₃, F, OCH₃, OCN, O(CH₂)[n]NH₂ orO(CH₂)[n]CH₃, where n is from 1 to about 10, and other substituentshaving similar properties.

Such oligonucleotides are best described as being functionallyinterchangeable with natural oligonucleotides or synthesizedoligonucleotides along natural lines, but which have one or moredifferences from natural structure. All such analogs are comprehended bythis invention so long as they function effectively to hybridize withmessenger RNA of CYP24 to inhibit the function of that RNA.

The oligonucleotides in accordance with this invention preferablycomprise from about 3 to about 100 subunits. It is more preferred thatsuch oligonucleotides and analogs comprise from about 8 to about 25subunits and still more preferred to have from about 12 to about 20subunits. As will be appreciated, a subunit is a base and sugarcombination suitably bound to adjacent subunits through phosphodiesteror other bonds. The oligonucleotides used in accordance with thisinvention may be conveniently and routinely made through the well-knowntechnique of solid phase synthesis. Equipment for such synthesis is soldby several vendors, including Applied Biosystems. Any other means forsuch synthesis may also be employed, however, the actual synthesis ofthe oligonucleotides is well within the talents of the routineer. Thepreparation of other oligonucleotides such as phosphorothioates andalkylated derivatives is also well known to those of skill in the art.

B) Ribozymes

In addition to antisense molecules, ribozymes can be used to target andinhibit transcription of CYP24. A ribozyme is an RNA molecule thatcatalytically cleaves other RNA molecules. Different kinds of ribozymeshave been described, including group I ribozymes, hammerhead ribozymes,hairpin ribozymes, RNAse P, and axhead ribozymes (see Castanotto et al.(1994) Adv. in Pharmacology 25: 289-317 for a general review of theproperties of different ribozymes).

The general features of hairpin ribozymes are described e.g., in Hampelet al. (1990) Nucl. Acids Res. 18: 299-304; Hampel et al. (1990)European Patent Publication No. 0 360 257; U.S. Pat. No. 5,254,678.Methods of preparing are well known to those of skill in the art (see,e.g., Wong-Staal et al., WO 94/26877; Ojwang et al. (1993) Proc. Natl.Acad. Sci. USA 90: 6340-6344; Yamada et al. (1994) Human Gene Therapy 1:39-45; Leavitt et al. (1995) Proc. Natl. Acad. Sci. USA 92: 699-703;Leavitt et al. (1994) Human Gene Therapy 5: 1151-120; and Yamada et al.(1994) Virology 205: 121-126).

C) Competitive Inhibition of CYP24 Polypeptide Activity.

CYP24 activity, e.g., at a given VDR activity level, can be decreased byprovision of a competitive inhibitor of the CYP24 polypeptide. This ismost simply accomplished by providing a CYP24 polypeptide that lacks25-hydroxyvitamin D3 24-hydroxylase enzyme activity.

Methods of making inactive polypeptide variants (muteins) are well knownto those of skill (see, e.g., U.S. Pat. Nos. 5,486,463, 5,422,260,5,116,943, 4,752,585, 4,518,504). Screening of such polypeptides (e.g.,in the assays described above) can be accomplished with only routineexperimentation. Using high-throughput methods, as described herein,literally thousands of agents can be screened in only a day or two.

D) Modification of Promoters to Regulate Endogenous CYP24 Expression.

In still another embodiment, the expression of CYP24 can be altered byaltering the endogenous promoter. Methods of altering expression ofendogenous genes are well known to those of skill in the art. Typicallysuch methods involve altering or replacing all or a portion of theregulatory sequences controlling expression of the particular gene thatis to be regulated. In a preferred embodiment, the regulatory sequences(e.g., the native promoter) upstream of the CYP24 gene is altered.

This is typically accomplished by the use of homologous recombination tointroduce a heterologous nucleic acid into the native regulatorysequences. To downregulate expression of the CYP24 gene product, simplemutations that either alter the reading frame or disrupt the promoterare suitable.

In a particularly preferred embodiment, nucleic acid sequencescomprising the structural gene in question or upstream sequences areutilized for targeting heterologous recombination constructs. The use ofhomologous recombination to alter expression of endogenous genes isdescribed in detail in U.S. Pat. No. 5,272,071, WO 91/09955, WO93/09222, WO 96/29411, WO 95/31560, and WO 91/12650.

E) Use of Other Molecules.

Numerous other approaches can be taken to downregulate CYP24 activity.As indicated above, particularly using high throughput screeningmethods, literally thousands of compounds can be tested for ability toalter (e.g. downregulate) CYP24 activity. Any one or more of thecompounds identified above or in such screening systems can be used tomodulate CYP24 activity.

F) Administration of CYP24 Modulators.

The compounds that modulate (e.g. downregulate) CYP24 activity can beadministered by a variety of methods including, but not limited toparenteral, topical, oral, or local administration, such as by aerosolor transdermally, for prophylactic and/or therapeutic treatment. Thepharmaceutical compositions can be administered in a variety of unitdosage forms depending upon the method of administration. For example,unit dosage forms suitable for oral administration include powder,tablets, pills, capsules and lozenges. It is recognized that the CYP24modulators (e.g. antibodies, antisense constructs, ribozymes, smallorganic molecules, etc.) when administered orally, must be protectedfrom digestion. This is typically accomplished either by complexing themolecule(s) with a composition to render it resistant to acidic andenzymatic hydrolysis or by packaging the molecule(s) in an appropriatelyresistant carrier such as a liposome. Means of protecting agents fromdigestion are well known in the art.

The compositions for administration will commonly comprise a CYP24modulator dissolved in a pharmaceutically acceptable carrier, preferablyan aqueous carrier. A variety of aqueous carriers can be used, e.g.,buffered saline and the like. These solutions are sterile and generallyfree of undesirable matter. These compositions may be sterilized byconventional, well known sterilization techniques. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents and the like, for example, sodiumacetate, sodium chloride, potassium chloride, calcium chloride, sodiumlactate and the like. The concentration of active agent in theseformulations can vary widely, and will be selected primarily based onfluid volumes, viscosities, body weight and the like in accordance withthe particular mode of administration selected and the patient's needs.

Thus, a typical pharmaceutical composition for intravenousadministration would be about 0.1 to 10 mg per patient per day. Dosagesfrom 0.1 up to about 100 mg per patient per day may be used,particularly when the drug is administered to a secluded site and notinto the blood stream, such as into a body cavity or into a lumen of anorgan. Substantially higher dosages are possible in topicaladministration. Actual methods for preparing parenterally administrablecompositions will be known or apparent to those skilled in the art andare described in more detail in such publications as Remington'sPharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa.(1980).

The compositions containing modulators of CYP24 can be administered fortherapeutic or prophylactic treatments. In therapeutic applications,compositions are administered to a patient suffering from a disease(e.g., an epithelial cancer) in an amount sufficient to cure or at leastpartially arrest the disease and its complications. An amount adequateto accomplish this is defined as a “therapeutically effective dose.”Amounts effective for this use will depend upon the severity of thedisease and the general state of the patient's health. Single ormultiple administrations of the compositions may be administereddepending on the dosage and frequency as required and tolerated by thepatient. In any event, the composition should provide a sufficientquantity of the agents of this invention to effectively treat thepatient.

VI. Kits for use in Diagnostic and/or Prognostic Applications.

For use in diagnostic, research, and therapeutic applications suggestedabove, kits are also provided by the invention. In the diagnostic andresearch applications such kits may include any or all of the following:assay reagents, buffers, CYP24 specific and/or VDR specific nucleicacids or antibodies (e.g. full-size monoclonal or polyclonal antibodies,single chain antibodies (e.g., scFv), or other CYP24 or VDR bindingmolecules), and other hybridization probes and/or primers, and/orsubstrates for 25-hydroxyvitamin D3 24-hydroxylase. A therapeuticproduct may include sterile saline or another pharmaceuticallyacceptable emulsion and suspension base.

In addition, the kits may include instructional materials containingdirections (i.e., protocols) for the practice of the methods of thisinvention. While the instructional materials typically comprise writtenor printed materials they are not limited to such. Any medium capable ofstoring such instructions and communicating them to an end user iscontemplated by this invention. Such media include, but are not limitedto electronic storage media (e.g., magnetic discs, tapes, cartridges,chips), optical media (e.g., CD ROM), and the like. Such media mayinclude addresses to internet sites that provide such instructionalmaterials.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Identification of CYP24 as a Driver Oncogene for Amplificationat 20q13.2

This experiment describes genetic analysis of breast tumors thatindicates selective amplification of CYP24. Selection for higher copynumber of this gene during tumor evolution provides further evidence ofthe importance of the vitamin D pathway in tumor development in thebreast.

We have used a new high resolution form of comparative genomichybridization, array CGH, to obtain a high resolution, quantitative mapof DNA copy number across a region of recurrent amplification atchromosome band 20q13.2. Array CGH, which was developed in ourlaboratories uses microarrays of DNA clones as the hybridization targetso that its resolution is determined by the spacing of the target clonesacross a genomic region (FIG. 1). Thus, when contiguous clones make upthe array, very high resolution copy number profiles can be obtained.

The unprecedented high dynamic range and quantitative accuracy of arrayCGH provides for the first time, the capability to very precisely mapcopy number profiles across an amplified region. In some tumors, copynumber profiles show narrow peaks of amplification (˜300 kb in FIG. 3).This information focuses attention on genes mapping to the region andindicates that they should be given highest priority for evaluation ascandidate driver oncogenes. The application of high resolution array CGHacross region A at chromosome band 20q13.2 in breast cancer revealed theexistence of two subregions, A1 and A2 with distinct amplificationbehavior. Recently a candidate oncogene, ZNF217 (Collins et al., 1998)has been identified that maps to subregion A1 and is likely to be thedriver gene for amplification of A1. Our attention is now focused on thegene CYP24 as the driver oncogene for region A2, because it maps to thenarrow genomic interval most highly amplified in these tumors.

Previously, CYP24 had been discounted as a candidate oncogene because itwas not found to be transcribed in the breast cancer cell line, BT474(Collins et al. (1998) Proc. Natl. Acad. Sci. USA 95: 8703-8708).However, re-evaluation of expression of CYP24 in cell lines and tumorswas warranted because of its position at the peak of the copy numberprofile and because of the existing knowledge of its function. Thereforewe examined expression levels of CYP24 and the vitamin D receptor (VDR),which controls CYP24 expression by RT-PCR using the primers listed inTable 1. This re-evaluation shows that these genes are expressed inbreast cancer cell lines and tumors (FIG. 3). Expression of CYP24 andVDR was detected in MCF7 cells and higher levels of expression of CYP24were induced when cells were treated with 1,25-dihydroxyvitamin-D3 (FIG.3A). Furthermore, expression of CYP24 and VDR was detected in two breasttumors S21 and S59 (FIG. 3B). In BT474 however, CYP24 expression was notdetected without addition of 1,25-dihydroxyvitamin-D3 to the culturemedium (FIG. 3C). Only low level expression of VDR was found in thiscell line, most likely accounting for the failure to detect expressionof CYP24 in BT474 without addition of 1,25-dihydroxyvitamin-D3. Theseobservations on BT474 illustrate the complexity of the analysis of CYP24function and emphasize the importance of measuring VDR activity whenevaluating the role of CYP24 in tumorigenesis.

TABLE 1 Primers used for assessing gene expression of CYP24 and VDR.SEQ ID Primer Name Sequence NO CYP24 Forward5′-(AAT TAA CCC TCA CTA AAG GG) CAA ACC GTG GAA 1 GGC CTA TC-3′*CYP24 Reverse 5′-(TAA TAC GAC TCA CTA TAG GGA G)T CTT CCC TTC 2CAG GAT CA-3′** VDR. Forward 5′-CTTCAGGCGAAGCATGAAGC-3′+ 3 VDR Reverse5′-CCTTCATCATGCCGATGTCC-3′ 4 ZNF2175′-(AAT TAA CCC TCA CTA AAG GG) AGA GGG GTG 5 Forward AGT GAC AAG-3′*ZNF217 5′-(TAA TAC GAC TCA CTA TAG GG) AGC TCG GAA TGG 6 ReverseAAC AAC-3′^(a) *T3 promoter shown in parentheses is included at the 5′end so that the amplified product can be used as a template for in vitrotranscription to generate riboprobes for mRNA FISH. **T7 promoter shownin parentheses is included at the 5′ end so that the amplified productcan be used as a template for in vitro transcription to generateriboprobes for mRNA FISH. The reverse primer spans the second exon-exonjunction, preventing amplification of genomic DNA. A 111 bp fragment isamplified. +Spans the third exon-exon junction. ++ A 134 bp PCR fragmentis amplified. ^(a)A 265 bp PCR fragment is amplified.

Example 2 Expression Analysis using Multi-Color Fluorescent in SituHybridization (mRNA FISH) on Tissue Sections

In order to identify genes that are overexpressed in tumor compared tonormal tissue, we have adapted our FISH protocols for visualizingtranscription patterns in C. elegans (Albertson et al. (1995) pages339-364 In C. elegans: Modern Biological Analysis of an Organism, vol.48, H. F. Epstein and D. C. Shakes, eds. Academic Press, Inc; Birchallet al. (1995) Nature Genet. 11: 314-320) for use with formalin fixedparaffin embedded clinical specimens. Our approach involves the use offluorescently labeled riboprobes that are synthesized by in vitrotranscription. The DNA template for the transcription reaction isgenerated by amplification using gene specific primers in which the T3or T7 phage promoter has been incorporated in the 5′ end. Thus,subcloning of gene fragments to make probes can be avoided.

The hybridization signal was imaged with a confocal microscope, thatreduces interference from tissue autofluorescence because of the narrowwavelength exciting light and the exclusion of out of focusfluorescence. The use of fluorescent probes, rather than radioactiveprobes has a number of advantages including, higher resolution, timesaving, compatability with simultaneous immunohistochemistry (Chuang etal. (1996) Cell, 79: 1-20) and the possibility of measuring relativelevels of expression of a number of genes simultaneously on a singletissue section (Albertson et al., 1995).

Example 3 Expression of CYP24 and VDR in Normal Mammary Cells

In the human, vitamin D receptors have been localized byimmunohistochemistry to the luminal and alveolar epithelial cells of thenormal breast and in breast tumor cells (Berger et al. (1987) CancerRes. 47: 6793-6799; Colston et al. (1989) Lancet, 188-191). In thisexperiment, the expression profiles of the CYP24 and VDR genes duringvarious stages of murine mammary gland development and involution aredetermined in order to identify the cell types and developmental stagesin which these gene products function. These studies will provide thedescription of the normal expression of these genes, which are thencompared to expression in murine breast tumor models and the CYP24transgenic mouse to be developed as described below.

The expression analysis is carried out at both the transcript andprotein levels. As described above gene specific probes for CYP24 andVDR mRNAs can be used to generate riboprobes for mRNA FISH. Antibodiesspecific for VDR are commercially available (Affinity BioReagents#PA1-711, MA1-710; Santa Cruz Biotechnology #sc-1008, sc-1009)

Immunohistochemistry and/or a combination of mRNA FISH andimmunohistochemistry are used to localize the site of expression ofparticular genes and marker proteins specific for various cell types inthe breast. Where possible, localization of expression of CYP24 and/orVDR and the cell type specific markers is carried out simultaneously onthe same tissue sections using multiple distinguishable fluorochromes onthe probes for the genes and marker proteins.

Development of the rodent breast has been described (see, e.g., Medina(1996) J. Mamm. Gland Biol. Neopl. 1: 5-19) and begins by arborizationof the ductal system throughout the mammary fat pad at 4-8 weeks of age.The terminal end-buds, located at the leading edge of the invading ductscontain proliferating cells. At the time of pregnancy, furtherarborization of the ductal system takes place by elaboration of tertiaryend-buds from the sides of the existing ductal tree. Terminaldifferentiation of the gland takes place during lactation when the milkproteins, lactoglobulin and whey acidic protein are synthesized.Involution of the lactating mammary gland involves extensive apoptosisand occurs during 4-6 weeks following weaning.

Experimental Design.

Expression of CYP24 and VDR is determined by in situ staining of tissuesections using mRNA FISH or gene specific antibodies. Tissue blocks areprepared following in vivo perfusion and fixation of the mice. Mammaryglands are harvested from mice at: (a) the beginning and end of breastductal arborization (at 3-4 weeks and at 8 weeks, respectively), (b) atearly, intermediate and late stages of pregnancy (at 4, 8, 13, and 18days post coitus), (c) during lactation, and (d) during early and latebreast involution (at 4 and 8 weeks after elective weaning). Prior tosacrifice, all mice are injected with 5-bromo-deoxyuridine (BrdU) forimmunohistochemical detection of S-phase cells using monoclonal BrdUantibody (Arbeit, et al., 1994). Immunohistochemical staining of keratinintermediate filament proteins is used to distinguish the basal(keratin-14) and luminal cells (keratin-6) of the ducts (Antibodies,BabCo #prb-155p, -169p). The early and late stages of involution isidentified by using the TUNEL assay for apoptosis (Naik et al. (1996)Genes Dev. 10: 2106-2166).

Methods.

Specimen Preparation.

Mice are injected i.p. with 100 mg/kg BrdU 2 hrs prior to sacrifice. Themice are weighed and anesthetized with 37.5 mg/kg of a 0.25% Avertinsolution and perfused with a 3.75% solution of freshly preparedparaformaldehyde. Tissues are removed, post-fixed overnight in 3.75%paraformaldehyde at 4° C. and then embedded in paraffin. Sections (˜6 μmthickness) are de-waxed in xylene, taken through a graded series ofethanols and then incubated with 5-15 μg/ml of proteinase K at 37° C.for 15 min., depending on the application. Following protease treatmentthe specimens are post-fixed in 1% paraformaldehyde for 20 min. at roomtemperature, rinsed and then dehydrated.

mRNA FISH.

Specimens are pre-hybridized in hybridization buffer (50-70% formamide,5×SSC, 0.1% SDS, 0.1% Tween 20, 100 μg/ml tRNA, 10% dextran sulfate) at37° C. for 2 hrs. The pre-hybridization solution is removed and thefluorescently labeled riboprobe (Albertson et al. (1995) pages 339-364In C. elegans: Modern Biological Analysis of an Organism, vol. 48, H. F.Epstein and D. C. Shakes, eds. Academic Press, Inc) are applied to thespecimen in hybridization buffer. Hybridization is carried out overnightat 37-50° C. depending on the length and GC content of the probe.

Immunohistochemistry.

Processing of sections varies slightly depending on the antibody, butwill use standard methods for indirect detection (e.g. Albertson (1984)Develop. Biol. 101: 61-72).

S Phase Analysis.

After immunohistochemical staining for BrdU positive cells, the BrdUlabeling index is determined by counting 1000 nuclei in sequential 20×fields.

Apoptosis.

The TUNEL assay is carried out using fluorescent detection of terminaltransferase activity according to the manufacturer's directions (Oncor#S7110, Gaiterburg, Md.).

Data Collection and Analysis.

Expression profiles of CYP24 and VDR in mammary tissue includesenumeration of specific cell types, developmental stage-specificexpression patterns and relative levels of expression. Expression ofcell specific marker proteins is used to confirm assignment of CYP24 andVDR expression in particular cells and developmental stages. The basaland luminal cells of the ducts are distinguished by their expression ofparticular keratins and proliferating terminal end-buds will beidentified by BrdU incorporation. Involuting cells are identified asTUNEL positive cells. The relative levels of expression of CYP24 and VDRmRNA at the different developmental stages are measured relative to aribosomal probe hybridized to the same tissue sections.

Example 4 Expression of CYP24 in the Established Murine Breast CancerModel, MMTV-ERBB2 Transgenic Mouse

We have documented the expression of CYP24 and VDR in human breast tumorspecimens (FIG. 3) and will continue to survey expression of these genesin normal and tumor tissue from human breast tumor specimens. Here, wewill investigate the expression of CYP24 and VDR during breastcarcinogenesis in an established transgenic mouse model of breastcancer, in which the ERBB2 oncogene is expressed in mammary tissue underthe control of the mouse mammary tumor virus promoter (JAX Mice, MMTVneuErbb2, #002376). These mice first develop focal tumors in hyperplastic,dysplastic mammary glands at ˜4 months (Guy et al., 1992). The study oftransgenic mouse models of breast carcinogenesis offers the opportunityto investigate the potential role of these genes in certain aspects oftumorigenesis that cannot be studied by analysis of patient material. Inparticular, mouse models offer access to premalignant stages generallynot available from human specimens. Furthermore, murine tumor modelsallow the role of particular genes in tumorigenesis to be assessed intumors induced in a defined genetic background (e.g. tumors induced byoverexpression of ERBB2, cyclin D1 or loss of p53).

Experimental Design.

Transgenic mice of 2, 4, 6 and 10-12 months are studied to encompasstime points of early and late tumor development. Two hours prior tosacrifice, BrdU is injected intra-peritoneally to measure S-phasekinetics. Tissues are harvested and processed for mRNA FISH, andexpression of keratins-14 and -6, and the HER2-neu transgene aredetermined, using antibodies as in Example 3.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. A method of detecting a breast cancer marker in an animal, said method comprising: detecting a level of CYP24 within a biological sample from said animal; comparing said level of CYP24 with a level of CYP24 in a control sample; and identifying the animal as one who has a breast cancer marker and is a candidate for a breast cancer therapy if an increased level of CYP24 in said biological sample compared to the level of CYP24 in said control sample is detected.
 2. The method of claim 1, wherein said level of CYP24 is detected by determining the copy number of CYP24 genes in the cells of said biological sample.
 3. The method of claim 2, wherein said copy number is measured using Comparative Genomic Hybridization (CGH).
 4. The method of claim 1, wherein said copy number is determined by hybridization to an array of nucleic acid probes.
 5. The method of claim 3, wherein said Comparative Genomic Hybridization is performed on an array.
 6. The method of claim 1, wherein said level of CYP24 is detected by measuring the level of CYP24 mRNA in said biological sample, wherein an increased level of CYP24 mRNA in said biological sample compared to CYP24 mRNA in said control sample indicates that the animal is one who has a breast cancer marker and is a candidate for a breast cancer therapy.
 7. The method of claim 6, wherein said biological and control samples have the same vitamin D receptor activity or the CYP24 mRNA levels are normalized to the levels of vitamin D receptor activity in said biological and control samples.
 8. The method of claim 6, wherein said level of CYP24 mRNA is measured by hybridization to one or more probes on an array.
 9. The method of claim 1, wherein said level of CYP24 is detected by measuring the level of CYP24 protein in said biological sample, wherein an increased level of CYP24 protein in said sample as compared to CYP24 protein in said control sample indicates that the animal is one who has a breast cancer marker and is a candidate for a breast cancer therapy.
 10. The method of claim 9, wherein said biological and control samples have the same vitamin D receptor activity or the protein levels are normalized to the levels of vitamin D receptor activity in said biological and control samples.
 11. The method of claim 1, wherein said level of CYP24 is detected by measuring the level of 25-hydroxyvitamin D3 24-hydroxylase enzyme activity in said biological sample, wherein an increased level of 25-hydroxyvitamin D3 24-hydroxylase enzyme activity in said sample as compared to 25-hydroxyvitamin D3 24-hydroxylase enzyme activity in said control sample indicates that the animal is one who has a breast cancer marker and is a candidate for a breast cancer therapy.
 12. The method of claim 11, wherein said biological and control samples have the same vitamin D receptor activity or the 25-hydroxyvitamin D3 24-hydroxylase activity levels are normalized to the level of vitamin D receptor activity in said biological and control samples.
 13. The method of claim 1, wherein said animal is a mammal selected from the group consisting of humans, non-human primates, canines, felines, murines, bovines, equines, porcines, and lagomorphs.
 14. The method of claim 1, wherein said biological sample is selected from the group consisting of excised tissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, and urine.
 15. The method of claim 1, wherein said increased level of CYP24 in said biological sample is at least about 2-fold greater than the level of CYP24 in said control sample.
 16. The method of claim 1, wherein said increased level of CYP24 in said biological sample is at least about 4-fold greater than said level of CYP24 in said control sample.
 17. A method of estimating the survival expectancy of an animal with breast cancer, said method comprising: detecting a level of CYP24 within a biological sample from said animal; comparing said level of CYP24 with the level of CYP24 in a control sample; and determining that said animal has a reduced survival expectancy compared to an animal with breast cancer that has a normal level of CYP24 if an increased level of CYP24 in said biological sample relative to the level of CYP24 in said control sample is detected.
 18. The method of claim 9, wherein the level of CYP24 protein is measured by immunoassay using at least one antibody that specifically binds to CYP24 protein.
 19. The method of claim 1, wherein the animal is suspected of having breast cancer.
 20. The method of claim 1, wherein said breast cancer marker is found to be present in said animal, and said method further comprises subjecting said human to an adjuvant cancer therapy. 